Single arm type I and type II receptor fusion proteins and uses thereof

ABSTRACT

In certain aspects, the disclosure provides soluble single-arm heteromeric polypeptide complexes comprising an extracellular domain of a type I serine/threonine kinase receptor of the TGF-beta family or an extracellular domain of a type II serine/threonine kinase receptor of the TGF-beta family. In some embodiments, the disclosure provides soluble single-arm polypeptide complexes comprising an extracellular domain of a type II receptor selected from: ActRIIA, ActRIIB, TGFBRII, BMPRII, and MISRII. In some embodiments, the disclosure provides soluble single-arm polypeptide complexes comprising an extracellular domain of a type I receptor selected from: ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7. Optionally the soluble complex is a heterodimer. In certain aspects, such soluble polypeptide complexes may be used for the treatment or prevention of various TGF-beta associated conditions, including without limitation diseases and disorders associated with, for example, cancer, muscle, bone, fat, red blood cells, metabolism, fibrosis and other tissues that are affected by one or more ligands of the TGF-beta superfamily.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 62/143,579, filed Apr. 6, 2015, and 62/259,422,filed Nov. 24, 2015. The disclosures of the foregoing applications arehereby incorporated by reference in their entirety

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 26, 2016, isnamed PHPH110101 SL.txt and is 443,689 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The superfamily isdivided into two general phylogenetic clades: the more recently evolvedmembers of the superfamily, which includes TGF-betas, activins, andnodal and the Glade of more distantly related proteins of thesuperfamily, which includes a number of BMPs and GDFs. Hinck (2012) FEBSLetters 586:1860-1870. TGF-beta superfamily members have diverse, oftencomplementary biological effects. By manipulating the activity of amember of the TGF-beta superfamily, it is often possible to causesignificant physiological changes in an organism. For example, thePiedmontese and Belgian Blue cattle breeds carry a loss-of-functionmutation in the GDF8 (also called myostatin) gene that causes a markedincrease in muscle mass. Grobet et al. (1997) Nat Genet., 17(1):71-4.Furthermore, in humans, inactive alleles of GDF8 are associated withincreased muscle mass and, reportedly, exceptional strength. Schuelke etal. (2004) N Engl J Med, 350:2682-8.

Changes in muscle, bone, fat, red blood cells, and other tissues may beachieved by enhancing or inhibiting signaling (e.g., SMAD 1, 2, 3, 5,and/or 8) that is mediated by ligands of the TGF-beta superfamily. Thus,there is a need for agents that regulate the activity of various ligandsof the TGF-beta superfamily.

SUMMARY OF THE INVENTION

In part, the disclosure provides heteromultimeric complexes comprising asingle TGF-beta superfamily type I or type II serine/threonine kinasereceptor polypeptide (e.g., an ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7,ActRIIA, ActRIIB, TGFBRII, BMPRII, or MISRII polypeptide), includingfragments and variants thereof. These constructs may be referred toherein as “single-arm” polypeptide complexes. Optionally, single-armpolypeptide complexes disclosed herein (e.g., a single-arm ActRIIBpolypeptide complex, such as an ActRIIB-Fc:Fc heterodimer) havedifferent ligand-binding specificities/profiles compared to acorresponding homodimeric complex (e.g., an ActRIIB homodimer, such asan ActRIIB-Fc:ActRIIB-Fc). Novel properties are exhibited byheteromultimeric polypeptide complexes comprising a single domain of aTGF-beta superfamily type I or type II serine/threonine kinase receptorpolypeptide, as shown by Examples herein.

Heteromultimeric structures include, for example, heterodimers,heterotrimers, and higher order complexes. Preferably, TGF-betasuperfamily type I and type II receptor polypeptides as described hereincomprise a ligand-binding domain of the receptor, for example, anextracellular domain of a TGF-beta superfamily type I or type IIreceptor. Accordingly, in certain aspects, protein complexes describedherein comprise an extracellular domain of a type II TGF-betasuperfamily receptor selected from: ActRIIA, ActRIIB, TGFBRII, BMPRII,and MISRII, as well as truncations and variants thereof, or anextracellular domain of a type I TGF-beta superfamily receptor selectedfrom: ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7, as well astruncations and variants thereof. Preferably, TGF-beta superfamily typeI and type II polypeptides as described herein, as well as proteincomplexes comprising the same, are soluble. In certain aspects,heteromultimer complexes of the disclosure bind to one or more TGF-betasuperfamily ligands (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6,BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8,GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A,activin B, activin C, activin E, activin AB, activin AC, activin AE,activin BC, activin BE, nodal, glial cell-derived neurotrophic factor(GDNF), neurturin, artemin, persephin, Müllerian-inhibiting substance(MIS), and Lefty). Optionally, protein complexes of the disclosure bindto one or more of these ligands with a K_(D) of less than or equal to10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹². In general, heteromultimer complexesof the disclosure antagonize (inhibit) one or more activities of atleast one TGF-beta superfamily ligand, and such alterations in activitymay be measured using various assays known in the art, including, forexample, a cell-based assay as described herein. Preferably, proteincomplexes of the disclosure exhibit a serum half-life of at least 4, 6,12, 24, 36, 48, or 72 hours in a mammal (e.g., a mouse or a human).Optionally, protein complexes of the disclosure may exhibit a serumhalf-life of at least 6, 8, 10, 12, 14, 20, 25, or 30 days in a mammal(e.g., a mouse or a human).

In certain aspects, protein complexes described herein comprise a firstpolypeptide covalently or non-covalently associated with a secondpolypeptide wherein the first polypeptide comprises the amino acidsequence of a TGF-beta superfamily type I or type II receptorpolypeptide and the amino acid sequence of a first member of aninteraction pair and the second polypeptide comprises a second member ofthe interaction pair and does not contain an amino acid sequence of aTGF-beta superfamily type I or type II receptor polypeptide. Optionally,the second polypeptide comprises, in addition to the second member ofthe interaction pair, a further polypeptide sequence that is not aTGF-beta superfamily type I or type II receptor polypeptide and mayoptionally comprise not more than 5, 10, 15, 20, 30, 40, 50, 100, 200,300, 400 or 500 amino acids. Optionally, the TGF-beta superfamily type Ior type II receptor polypeptide is connected directly to the firstmember of the interaction pair, or an intervening sequence, such as alinker, may be positioned between the amino acid sequence of theTGF-beta superfamily type I or type II receptor polypeptide and theamino acid sequence of the first member of the interaction pair.Examples of linkers include, but are not limited to, the sequences TGGG(SEQ ID NO: 62), TGGGG (SEQ ID NO: 60), SGGGG (SEQ ID NO: 61), GGGGS(SEQ ID NO: 510), and GGG (SEQ ID NO: 58).

Interaction pairs described herein are designed to promote dimerizationor form higher order multimers. In some embodiments, the interactionpair may be any two polypeptide sequences that interact to form acomplex, particularly a heterodimeric complex although operativeembodiments may also employ an interaction pair that forms a homodimericcomplex. The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate. Accordingly, firstand second members of an asymmetric interaction pair may associate toform a heterodimeric complex. Alternatively, the interaction pair may beunguided, meaning that the members of the pair may associate with eachother or self-associate without substantial preference and thus may havethe same or different amino acid sequences. Accordingly, first andsecond members of an unguided interaction pair may associate to form ahomodimer complex or a heterodimeric complex. Optionally, the firstmember of the interaction pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates covalently with the second member of theinteraction pair. Optionally, the first member of the interaction pair(e.g., an asymmetric pair or an unguided interaction pair) associatesnon-covalently with the second member of the interaction pair.

Traditional Fc fusion proteins and antibodies are examples of unguidedinteraction pairs, whereas a variety of engineered Fc domains have beendesigned as asymmetric interaction pairs. Therefore, a first memberand/or a second member of an interaction pair described herein maycomprise a constant domain of an immunoglobulin, including, for example,the Fc portion of an immunoglobulin. Optionally, a first member of aninteraction pair may comprise an amino acid sequence that is derivedfrom an Fc domain of an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. Forexample, the first member of an interaction pair may comprise, consistessentially of, or consist of an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to any one of SEQ ID NOs: 200-214. Optionally, a second memberof an interaction pair may comprise an amino acid sequence that isderived from an Fc domain of an IgG1, IgG2, IgG3, or IgG4. For example,the second member of an interaction pair may comprise, consistessentially of, or consist of an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to any one of SEQ ID NOs: 200-214. In some embodiments, afirst member and a second member of an interaction pair comprise Fcdomains derived from the same immunoglobulin class and subtype. In otherembodiments, a first member and a second member of an interaction paircomprise Fc domains derived from different immunoglobulin classes orsubtypes. Optionally, a first member and/or a second member of aninteraction pair (e.g., an asymmetric pair or an unguided interactionpair) comprise a modified constant domain of an immunoglobulin,including, for example, a modified Fc portion of an immunoglobulin. Forexample, protein complexes of the disclosure may comprise a first Fcportion of an IgG comprising an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto an amino acid sequence selected from the group: SEQ ID NOs: 200-214and a second Fc portion of an IgG, which may be the same or differentfrom the amino acid sequence of the first modified Fc portion of theIgG, comprising an amino acid sequence that is at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence selected from the group: SEQ ID NOs: 200-214.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a single type I or type II TGF-beta superfamilyreceptor polypeptide, wherein the TGF-beta superfamily receptorpolypeptide is derived from an ActRIIA receptor. For example, ActRIIApolypeptides may comprise, consist essentially of, or consist of anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to an ActRIIA sequence disclosed herein (e.g., SEQID NOs: 9, 10, 11, 101, 103, 401, and 402). Optionally, ActRIIApolypeptides may comprise, consist essentially of, or consist of anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to a polypeptide that a) begins at any one ofamino acids of 21-30 (e.g., amino acid residues 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) SEQ ID NO: 9, and b) ends at any one of amino acids110-135 (e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or135) of SEQ ID NO: 9. Optionally, ActRIIA polypeptides of the disclosuremay be fusion proteins that further comprise one or more portions(domains) that are heterologous to ActRIIA. For example, an ActRIIApolypeptide may be fused to a heterologous polypeptide that comprises amultimerization domain, optionally with a linker domain positionedbetween the ActRIIA polypeptide and the heterologous polypeptide (e.g.,SEQ ID NOs: 101, 103, 401, and 402). In some embodiments,multimerization domains described herein comprise one component of aninteraction pair. Heteromeric complexes that comprise an ActRIIApolypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ActRIIB receptor. For example, ActRIIB polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ActRIIB sequence disclosed herein (e.g., SEQ ID NOs: 1,2, 3, 4, 5, 6, 104, 106, 403, and 404). Optionally, ActRIIB polypeptidesmay comprise, consist essentially of, or consist of an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or29) SEQ ID NO: 1, and b) ends at any one of amino acids 109-134 (e.g.,amino acid residues 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, or 134 of SEQ ID NO: 1. Optionally, ActRIIB polypeptides of thedisclosure may be fusion proteins that further comprise one or moreportions (domains) that are heterologous to ActRIIB For example, anActRIIB polypeptide may be fused to a heterologous polypeptide thatcomprises a multimerization domain, optionally with a linker domainpositioned between the ActRIIB polypeptide and the heterologouspolypeptide (e.g., SEQ ID NOs: 104, 106, 403, and 404). In someembodiments, multimerization domains described herein comprise onecomponent of an interaction pair. Heteromeric complexes that comprise anActRIIB polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from a TGFBRII receptor. For example, TGFBRII polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an TGFBRII sequence disclosed herein (e.g., SEQ ID NOs: 42,43, 67, 68, 113, 115, 409, and 410). Optionally, TGFBRII polypeptidesmay comprise, consist essentially of, or consist of an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 of SEQ ID NO: 42, and b)ends at any one of amino acids 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165 or 166 of SEQ ID NO: 42. Optionally, TGFBRII polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43 or 44 of SEQ ID NO: 67, and b) ends at any one of amino acids163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190 or191 of SEQ ID NO: 67. Optionally, TGFBRII polypeptides of the disclosuremay be fusion proteins that further comprise one or more portions(domains) that are heterologous to TGFBRII. For example, a TGFBRIIpolypeptide may be fused to a heterologous polypeptide that comprises amultimerization domain, optionally with a linker domain positionedbetween the TGFBRII polypeptide and the heterologous polypeptide (e.g.,SEQ ID NOs: 113, 115, 409, and 410). In some embodiments,multimerization domains described herein comprise one component of aninteraction pair. Heteromeric complexes that comprise a TGFBRIIpolypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from a BMPRII receptor. For example, BMPRII polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a BMPRII sequence disclosed herein (e.g., SEQ ID NOs: 46,47, 71, 72, 107, 109, 405, and 406). Optionally, BMPRII polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of27-34 (e.g., amino acid residues 27, 28, 29, 30, 31, 32, 33, and 34) SEQID NO: 46 or 71, and b) ends at any one of amino acids 123-150 (e.g.,amino acid residues 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, and 150) of SEQ ID NO: 46 or 71. Optionally, BMPRIIpolypeptides of the disclosure may be fusion proteins that furthercomprise one or more portions (domains) that are heterologous to BMPRII.For example, a BMPRII polypeptide may be fused to a heterologouspolypeptide that comprises a multimerization domain, optionally with alinker domain positioned between the BMPRII polypeptide and theheterologous polypeptide (e.g., SEQ ID NOs: 107, 109, 405, and 406).Heteromeric complexes that comprise a BMPRII polypeptide do not compriseanother type I or type II TGF-beta superfamily receptor polypeptide butmay contain additional polypeptides that are not type I or type IITGF-beta superfamily receptor polypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an MISRII receptor. For example, MISRII polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MISRII sequence disclosed herein (e.g., SEQ ID NOs: 50,51, 75, 76, 79, 80, 110, 112, 407, and 408). Optionally, MISRIIpolypeptides may comprise, consist essentially of, or consist of anamino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to a polypeptide that a) begins at any one ofamino acids of 17-24 (e.g., amino acid residues 17, 18, 19, 20, 21, 22,23, and 24) SEQ ID NO: 50, 75, or 79, and b) ends at any one of aminoacids 116-149 (e.g., amino acid residues 116, 117, 118, 119, 120, 121,122 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, and149) of SEQ ID NO: 50, 75, or 79. Optionally, MISRII polypeptides of thedisclosure may be fusion proteins that further comprise one or moreportions (domains) that are heterologous to MISRII. For example, anMISRII polypeptide may be fused to a heterologous polypeptide thatcomprises a multimerization domain, optionally with a linker domainpositioned between the MISRII polypeptide and the heterologouspolypeptide (e.g., SEQ ID NOs: 110, 112, 407, and 408). In someembodiments, multimerization domains described herein comprise onecomponent of an interaction pair. Heteromeric complexes that comprise anMISRII polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK1 receptor. For example, ALK1 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK1 sequence disclosed herein (e.g., SEQ ID NOs: 14,15, 116, 118, 411, and 412). Optionally, ALK1 polypeptides may comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to apolypeptide that a) begins at any one of amino acids of 22-34 (e.g.,amino acid residues 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and34) SEQ ID NO: 14, and b) ends at any one of amino acids 95-118 (e.g.,amino acid residues 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, and 118) ofSEQ ID NO: 14. Optionally, ALK1 polypeptides of the disclosure may befusion proteins that further comprise one or more portions (domains)that are heterologous to ALK1. For example, an ALK1 polypeptide may befused to a heterologous polypeptide that comprises a multimerizationdomain, optionally with a linker domain positioned between the ALK1polypeptide and the heterologous polypeptide (e.g., SEQ ID NOs: 116,118, 411, and 412). Heteromeric complexes that comprise an ALK1polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK2 receptor. For example, ALK2 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK2 sequence disclosed herein (e.g., SEQ ID NOs: 18,19, 119, 121, 413, and 414). Optionally, ALK2 polypeptides may comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to apolypeptide that a) begins at any one of amino acids of 21-35 (e.g.,amino acid residues 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, and 35) SEQ ID NO: 18, and b) ends at any one of amino acids 99-123(e.g., amino acid residues 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, and 123) of SEQ ID NO: 18. Optionally, ALK2 polypeptides of thedisclosure may be fusion proteins that further comprise one or moreportions (domains) that are heterologous to ALK2. For example, an ALK2polypeptide may be fused to a heterologous polypeptide that comprises amultimerization domain, optionally with a linker domain positionedbetween the ALK2 polypeptide and the heterologous polypeptide (e.g., SEQID NOs: 119, 121, 413, and 414). Heteromeric complexes that comprise anALK2 polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK3 receptor. For example, ALK3 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK3 sequence disclosed herein (e.g., SEQ ID NOs: 22,23, 122, 124, 415, and 416). Optionally, ALK3 polypeptides may comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to apolypeptide that a) begins at any one of amino acids of 24-61 (e.g.,amino acid residues 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, and 61) SEQ ID NO: 22, and b) ends at any one ofamino acids 130-152 (e.g., amino acid residues 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, and 152) of SEQ ID NO: 22. Optionally, ALK3 polypeptidesof the disclosure may be fusion proteins that further comprise one ormore portions (domains) that are heterologous to ALK3. For example, anALK3 polypeptide may be fused to a heterologous polypeptide thatcomprises a multimerization domain, optionally with a linker domainpositioned between the ALK3 polypeptide and the heterologous polypeptide(e.g., SEQ ID NOs: 122, 124, 415, and 416). Heteromeric complexes thatcomprise an ALK3 polypeptide do not comprise another type I or type IITGF-beta superfamily receptor polypeptide but may contain additionalpolypeptides that are not type I or type II TGF-beta superfamilyreceptor polypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK4 receptor. For example, ALK4 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK4 sequence disclosed herein (e.g., SEQ ID NOs: 26,27, 83, 84, 125, 127, 417, and 418). Optionally, ALK4 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of23-34 (e.g., amino acid residues 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34) SEQ ID NO: 26 or 83, and b) ends at any one of amino acids101-126 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, and 126) of SEQ ID NO: 26 or 83. Optionally, ALK4polypeptides of the disclosure may be fusion proteins that furthercomprise one or more portions (domains) that are heterologous to ALK4.For example, an ALK4 polypeptide may be fused to a heterologouspolypeptide that comprises a multimerization domain, optionally with alinker domain positioned between the ALK4 polypeptide and theheterologous polypeptide (e.g., SEQ ID NOs: 125, 127, 417, and 418).Heteromeric complexes that comprise an ALK4 polypeptide do not compriseanother type I or type II TGF-beta superfamily receptor polypeptide butmay contain additional polypeptides that are not type I or type IITGF-beta superfamily receptor polypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK5 receptor. For example, ALK5 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK5 sequence disclosed herein (e.g., SEQ ID NOs: 30,31, 87, 88, 128, 130, 419, and 420). Optionally, ALK5 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, and 36) SEQ ID NO: 30 or 87, and b) ends at any one of amino acids106-126 (e.g., amino acid residues 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and126) of SEQ ID NO: 30 or 87. Optionally, ALK5 polypeptides of thedisclosure may be fusion proteins that further comprise one or moreportions (domains) that are heterologous to ALK5. For example, an ALK5polypeptide may be fused to a heterologous polypeptide that comprises amultimerization domain, optionally with a linker domain positionedbetween the ALK5 polypeptide and the heterologous polypeptide (e.g., SEQID NOs: 128, 130, 419, and 420). Heteromeric complexes that comprise anALK5 polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK6 receptor. For example, ALK6 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK6 sequence disclosed herein (e.g., SEQ ID NOs: 34,35, 91, 92, 131, 133, 421, and 422). Optionally, ALK6 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a polypeptide that a) begins at any one of amino acids of14-32 (e.g., amino acid residues 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, and 32) SEQ ID NO: 34, and b) ends atany one of amino acids 102-126 (e.g., amino acid residues 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, and 126) of SEQ ID NO: 34.Optionally, ALK6 polypeptides may comprise, consist essentially of, orconsist of an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to a polypeptide that a) begins atany one of amino acids of 26-62 (e.g., amino acid residues 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62) SEQ IDNO: 91, and b) ends at any one of amino acids 132-156 (e.g., amino acidresidues 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, and 156) ofSEQ ID NO: 91. Optionally, ALK6 polypeptides of the disclosure may befusion proteins that further comprise one or more portions (domains)that are heterologous to ALK6. For example, an ALK6 polypeptide may befused to a heterologous polypeptide that comprises a multimerizationdomain, optionally with a linker domain positioned between the ALK6polypeptide and the heterologous polypeptide (e.g., SEQ ID NOs: 131,133, 421, and 422). Heteromeric complexes that comprise an ALK6polypeptide do not comprise another type I or type II TGF-betasuperfamily receptor polypeptide but may contain additional polypeptidesthat are not type I or type II TGF-beta superfamily receptorpolypeptides.

In some embodiments, the disclosure provides heteromeric polypeptidecomplexes comprising a type I or type II TGF-beta superfamily receptorpolypeptide, wherein the TGF-beta superfamily receptor polypeptide isderived from an ALK7 receptor. For example, ALK7 polypeptides maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an ALK7 sequence disclosed herein (e.g., SEQ ID NOs: 38,39, 134, 136, 301, 302, 305, 306, 309, 310, 313, 423, and 424).Optionally, ALK7 polypeptides may comprise, consist essentially of, orconsist of an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to a polypeptide that begins at anyone of amino acids 21-28 of SEQ ID NO: 38 (e.g., amino acids 21, 22, 23,24, 25, 26, 27, or 28) and ends at any one of amino acids 92-113 of SEQID NO: 38 (e.g., amino acids 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 of SEQ IDNO: 38). Optionally, ALK7 polypeptides of the disclosure may be fusionproteins that further comprise one or more portions (domains) that areheterologous to ALK7. For example, an ALK7 polypeptide may be fused to aheterologous polypeptide that comprises a multimerization domain,optionally with a linker domain positioned between the ALK7 polypeptideand the heterologous polypeptide (e.g., SEQ ID NOs: 134, 136, 423, and424). Heteromeric complexes that comprise an ALK7 polypeptide do notcomprise another type I or type II TGF-beta superfamily receptorpolypeptide but may contain additional polypeptides that are not type Ior type II TGF-beta superfamily receptor polypeptides.

In some embodiments, the TGF-beta superfamily type I and/or type IIreceptor polypeptides disclosed herein comprise one or more modifiedamino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.In some embodiments, the TGF-beta superfamily type I and/or type IIpolypeptides described herein are glycosylated and have a glycosylationpattern obtainable from the expression of the polypeptides in amammalian cell, including, for example, a CHO cell.

In certain aspects the disclosure provides nucleic acids encoding any ofthe TGF-beta superfamily type I and/or type II polypeptides describedherein, including any fusion proteins comprising members of aninteraction pair. Nucleic acids disclosed herein may be operably linkedto a promoter for expression, and the disclosure further provides cellstransformed with such recombinant polynucleotides. Preferably the cellis a mammalian cell such as a COS cell or a CHO cell.

In certain aspects, the disclosure provides methods for making any ofthe TGF-beta superfamily type I and/or type II polypeptides describedherein as well as protein complexes comprising such a polypeptide. Sucha method may include expressing any of the nucleic acids disclosedherein in a suitable cell (e.g., CHO cell or a COS cell). Such a methodmay comprise: a) culturing a cell under conditions suitable forexpression of a TGF-beta superfamily type I or type II polypeptidesdescribed herein, wherein said cell is transformed with a type I or typeII polypeptide expression construct; and b) recovering the type I ortype II polypeptides so expressed. TGF-beta superfamily type I and/ortype II polypeptides described herein, as well as protein complexes ofthe same, may be recovered as crude, partially purified, or highlypurified fractions using any of the well-known techniques for obtainingprotein from cell cultures.

Any of the protein complexes described herein may be incorporated into apharmaceutical preparation. Optionally, such pharmaceutical preparationsare at least 80%, 85%, 90%, 95%, 97%, 98% or 99% pure with respect toother polypeptide components. Optionally, pharmaceutical preparationsdisclosed herein may comprise one or more additional active agents.

The disclosure further provides methods for use of the protein complexesand pharmaceutical preparations described herein for the treatment orprevention of various TGF-beta associated conditions, including withoutlimitation diseases and disorders associated with, for example, cancer,muscle, bone, fat, red blood cells, metabolism, fibrosis and othertissues that are affected by one or more ligands of the TGF-betasuperfamily.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows schematic examples of single-arm heteromeric proteincomplexes comprising either a type I receptor polypeptide or a type IIreceptor polypeptide. Such complexes can be assembled covalently ornoncovalently via a multimerization domain contained within eachpolypeptide chain. Two assembled multimerization domains constitute aninteraction pair, which can be either guided or unguided.

FIG. 2 shows a schematic example of a single-arm heteromeric proteincomplex comprising a type I receptor polypeptide (indicated as “I”)(e.g. a polypeptide that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to an extracellular domain of an ALK1, ALK2, ALK3,ALK4, ALK5, ALK6 or ALK7 protein from humans or other species) or a typeII receptor polypeptide (indicated as “II”) (e.g. a polypeptide that isat least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to anextracellular domain of an ActRIIA, ActRIIB, MISRII, BMPRII, or TGFBRIIprotein from humans or other species). In the illustrated embodiment,the type I or type II receptor polypeptide is part of a fusionpolypeptide that comprises a first member of an interaction pair (“B”),which associates with a second member of an interaction pair (“C”). Inthe fusion polypeptide, a linker may be positioned between the type I ortype II receptor polypeptide and the corresponding member of theinteraction pair. The first and second members of the interaction pair(B, C) may be a guided (asymmetric) pair, meaning that the members ofthe pair associate preferentially with each other rather thanself-associate, or the interaction pair may be unguided, meaning thatthe members of the pair may associate with each other or self-associatewithout substantial preference and may have the same or different aminoacid sequences. Traditional Fc fusion proteins and antibodies areexamples of unguided interaction pairs, whereas a variety of engineeredFc domains have been designed as guided (asymmetric) interaction pairs.

FIG. 3 shows an alignment of extracellular domains of human ActRIIA (SEQID NO: 500) and human ActRIIB (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 4 shows a multiple sequence alignment of various vertebrate ActRIIBprecursor proteins without their intracellular domains (SEQ ID NOs: 501,502, 503, 504, 505, and 506, respectively) human ActRIIA precursorprotein without its intracellular domain (SEQ ID NO: 507), and aconsensus ActRII precursor protein (SEQ ID NO: 508).

FIG. 5 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1. Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc to promote asymmetric chain pairing and the correspondingpositions with respect to other isotypes IgG2, IgG3 and IgG4. FIG. 5discloses SEQ ID NOS 208, 212, 209 and 210, respectively, in order ofappearance.

FIG. 6 shows ligand binding data for a single-arm ActRIIB-Fc:Fcheterodimeric protein complex compared to ActRIIB-Fc homodimer. For eachprotein complex, ligands are ranked by off-rate (k_(off) or k_(d)), akinetic constant that correlates well with ligand signaling inhibition,and listed in descending order of binding affinity (ligands bound mosttightly are listed at the top). At left, yellow, red, green, and bluelines indicate magnitude of the off-rate constant. Ligands of particularinterest are highlighted in bold while others are represented in gray,and solid black lines indicate ligands whose binding to heterodimer isenhanced or unchanged compared with homodimer, whereas dashed linesindicate substantially reduced binding compared with homodimer. Asshown, ActRIIB-Fc homodimer binds to each of five high affinity ligandswith similarly high affinity, whereas single-arm ActRIIB-Fcdiscriminates more readily among these ligands. Thus, single-armActRIIB-Fc binds strongly to activin B and GDF11 and with intermediatestrength to GDF8 and activin A. In further contrast to ActRIIB-Fchomodimer, single-arm ActRIIB-Fc displays only weak binding to BMP10 andno binding to BMP9. These data indicate that single-arm ActRIIB-Fc hasgreater ligand selectivity than homodimeric ActRIIB-Fc.

FIG. 7 shows ligand binding data for a single-arm ALK3-Fc:Fcheterodimeric protein complex compared to ALK3-Fc homodimer. Format isthe same as for FIG. 6. As shown, single-arm ALK3-Fc heterodimer retainsthe exceptionally tight binding to BMP4 observed with ALK3-Fc homodimer,whereas it exhibits reduced strength of binding to BMP2 and thereforediscriminates better between BMP4 and BMP2 than does ALK3-Fc homodimer.Single-arm ALK3-Fc also discriminates better among BM'S (intermediatebinding), GDF7 (weak binding), and GDF6 (no binding) compared to ALK3-Fchomodimer, which binds these three ligands with very similar strength(all intermediate). These data indicate that single-arm ALK3-Fc hasgreater ligand selectivity than homodimeric ALK3-Fc.

FIG. 8 shows ligand binding data for a single-arm ActRIIA-Fc:Fcheterodimeric protein complex compared to ActRIIA-Fc homodimer. Formatis the same as for FIG. 6. As shown, ActRIIA-Fc homodimer exhibitspreferential binding to activin B combined with strong binding toactivin A and GDF11, whereas single-arm ActRIIA-Fc has a reversedpreference for activin A over activin B combined with greatly enhancedselectivity for activin A over GDF11 (weak binder). These data indicatethat single-arm ActRIIA-Fc has substantially different ligandselectivity than homodimeric ActRIIA-Fc.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

In part, the present disclosure relates to single-arm heteromultimercomplexes comprising an extracellular domain of a TGFβ superfamily typeI receptor polypeptide or an extracellular domain of a TGFβ superfamilytype II receptor polypeptide, methods of making such single-armheteromultimer complexes, and uses thereof. As described herein,single-arm heteromultimer complexes may comprise an extracellular domainof a TGFβ superfamily type I receptor polypeptide selected from: ALK1,ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7, or an extracellular domain of aTGFβ superfamily type II receptor polypeptide selected from: ActRIIA,ActRIIB, TGFBRII, BMPRII, and MISRII. In certain preferred embodiments,heteromultimer complexes of the disclosure have an altered profile ofbinding to TGFβ superfamily ligands relative to a correspondinghomomultimer complex (e.g., an ActRIIB-Fc:Fc heterodimer compared to anActRIIB-Fc:ActRIIB-Fc homodimer complex).

The TGF-β superfamily is comprised of over thirty secreted factorsincluding TGF-betas, activins, nodals, bone morphogenetic proteins(BMPs), growth and differentiation factors (GDFs), and anti-Mullerianhormone (AMH). See, e.g., Weiss et al. (2013) Developmental Biology,2(1): 47-63. Members of the superfamily, which are found in bothvertebrates and invertebrates, are ubiquitously expressed in diversetissues and function during the earliest stages of developmentthroughout the lifetime of an animal. Indeed, TGF-β superfamily proteinsare key mediators of stem cell self-renewal, gastrulation,differentiation, organ morphogenesis, and adult tissue homeostasis.Consistent with this ubiquitous activity, aberrant TGF-beta superfamilysignaling is associated with a wide range of human pathologiesincluding, for example, autoimmune disease, cardiovascular disease,fibrotic disease, and cancer.

Ligands of the TGF-beta superfamily share the same dimeric structure inwhich the central 3-½ turn helix of one monomer packs against theconcave surface formed by the beta-strands of the other monomer. Themajority of TGF-beta family members are further stabilized by anintermolecular disulfide bond. This disulfide bonds traverses through aring formed by two other disulfide bonds generating what has been termeda ‘cysteine knot’ motif. See, e.g., Lin et al., (2006) Reproduction 132:179-190 and Hinck (2012) FEBS Letters 586: 1860-1870.

TGF-beta superfamily signaling is mediated by heteromeric complexes oftype I and type II serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins1, 2, 3, 5, and 8) upon ligand stimulation. See, e.g., Massagué (2000)Nat. Rev. Mol. Cell Biol. 1:169-178. These type I and type II receptorsare transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase specificity.In general, type I receptors mediate intracellular signaling while thetype II receptors are required for binding TGF-beta superfamily ligands.Type I and II receptors form a stable complex after ligand binding,resulting in phosphorylation of type I receptors by type II receptors.

The TGF-beta family can be divided into two phylogenetic branches basedon the type I receptors they bind and the Smad proteins they activate.One is the more recently evolved branch, which includes, e.g., theTGF-betas, activins, GDF8, GDF9, GDF11, BMP3 and nodal. The other branchcomprises the more distantly related proteins of the superfamily andincludes, e.g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10,GDF1, GDF5, GDF6, and GDF7. See, e.g. Hinck (2012) FEBS Letters586:1860-1870.

TGF-beta isoforms are the founding members of the TGF-beta superfamily,of which there are 3 known isoforms in mammals designated as TGF-beta1,TGF-beta2 and TGF-beta3. Mature bioactive TGF-beta ligands function ashomodimers and predominantly signal through the type I receptor ALK5 buthave also been found to signal through ALK1 in endothelial cells. See,e.g., Goumans et al. (2003) Mol Cell 12(4): 817-828. TGF-beta1 is themost abundant and ubiquitously expressed isoform. TGF-beta1 is known tohave an important role in wound healing, and mice expressing aconstitutively active TGF-beta1 transgene develop fibrosis. See e.g.,Clouthier et al., (1997) J Clin. Invest. 100(11): 2697-2713. TGF-beta1is also involved in T cell activation and maintenance of T regulatorycells. See, e.g., Li et al., (2006) Immunity 25(3): 455-471. TGF-beta2expression was first described in human glioblastoma cells and occurs inneurons and astroglial cells of the embryonic nervous system. TGF-beta2is also known to suppress interleukin-2-dependent growth of Tlymphocytes. TGF-beta3 was initially isolated from a humanrhabdomyosarcoma cell line and since has been found in lungadenocarcinoma and kidney carcinoma cell lines. TGF-beta3 is known to beimportant for palate and lung morphogenesis. See, e.g., Kubiczkova etal., (2012) Journal of Translational Medicine 10:183.

Activins are members of the TGF-beta superfamily that were initiallydiscovered as regulators of follicle-stimulating hormone secretion, butsubsequently various reproductive and non-reproductive roles have beencharacterized. Principal activin forms A, B, and AB arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B), respectively). The human genome also encodesan activin C and an activin E, which are primarily expressed in theliver, and heterodimeric forms containing β_(C) or β_(E) are also known.In the TGF-beta superfamily, activins are unique and multifunctionalfactors that can stimulate hormone production in ovarian and placentalcells, support neuronal cell survival, influence cell-cycle progresspositively or negatively depending on cell type, and induce mesodermaldifferentiation at least in amphibian embryos. See, e.g., DePaolo et al.(1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997) Curr Biol.7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963. In severaltissues, activin signaling is antagonized by its related heterodimer,inhibin. For example, in the regulation of follicle-stimulating hormone(FSH) secretion from the pituitary, activin promotes FSH synthesis andsecretion, while inhibin reduces FSH synthesis and secretion. Otherproteins that may regulate activin bioactivity and/or bind to activininclude follistatin (FS), follistatin-related protein (FSRP, also knownas FLRG or FSTL3), and α₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents thatspecifically bind to the β_(A) subunit, whether in the context of anisolated β_(A) subunit or as a dimeric complex (e.g., a β_(A)β_(A)homodimer or a β_(A)β_(B) heterodimer). In the case of a heterodimercomplex (e.g., a β_(A)β_(B) heterodimer), agents that bind to “activinA” are specific for epitopes present within the PA subunit, but do notbind to epitopes present within the non-PA subunit of the complex (e.g.,the β_(B) subunit of the complex). Similarly, agents disclosed hereinthat antagonize (inhibit) “activin A” are agents that inhibit one ormore activities as mediated by a β_(A) subunit, whether in the contextof an isolated β_(A) subunit or as a dimeric complex (e.g., a β_(A)β_(A)homodimer or a β_(A)β_(B) heterodimer). In the case of β_(A)β_(B)heterodimers, agents that inhibit “activin A” are agents thatspecifically inhibit one or more activities of the PA subunit but do notinhibit the activity of the non-β_(A) subunit of the complex (e.g., theβ_(B) subunit of the complex). This principle applies also to agentsthat bind to and/or inhibit “activin B”, “activin C”, and “activin E”.Agents disclosed herein that antagonize “activin AB” are agents thatinhibit one or more activities as mediated by the β_(A) subunit and oneor more activities as mediated by the β_(B) subunit.

The BMPs and GDFs together form a family of cysteine-knot cytokinessharing the characteristic fold of the TGF-beta superfamily. See, e.g.,Rider et al. (2010) Biochem J., 429(1):1-12. This family includes, forexample, BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known asGDF10), BMP4, BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known asGDF2), BMP10, BMP11 (also known as GDF11), BMP12 (also known as GDF7),BMP13 (also known as GDF6), BMP14 (also known as GDF5), BMP15, GDF1,GDF3 (also known as VGR2), GDF8 (also known as myostatin), GDF9, GDF15,and decapentaplegic. Besides the ability to induce bone formation, whichgave the BMPs their name, the BMP/GDFs display morphogenetic activitiesin the development of a wide range of tissues. BMP/GDF homo- andhetero-dimers interact with combinations of type I and type II receptordimers to produce multiple possible signaling complexes, leading to theactivation of one of two competing sets of SMAD transcription factors.BMP/GDFs have highly specific and localized functions. These areregulated in a number of ways, including the developmental restrictionof BMP/GDF expression and through the secretion of several proteins thatbind certain TGF-beta superfamily ligands with high affinity and therebyinhibit ligand activity. Curiously, some of these endogenous antagonistsresemble TGF-beta superfamily ligands themselves.

Growth and differentiation factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass and is highlyexpressed in developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of skeletal muscle. See, e.g., McPherron et al., Nature(1997) 387:83-90. Similar increases in skeletal muscle mass are evidentin naturally occurring mutations of GDF8 in cattle and, strikingly, inhumans. See, e.g., Ashmore et al. (1974) Growth, 38:501-507; Swatlandand Kieffer, J. Anim. Sci. (1994) 38:752-757; McPherron and Lee, Proc.Natl. Acad. Sci. USA (1997) 94:12457-12461; Kambadur et al., Genome Res.(1997) 7:910-915; and Schuelke et al. (2004) N Engl J Med, 350:2682-8.Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression. See, e.g., Gonzalez-Cadavid et al., PNAS (1998) 95:14938-43.In addition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation. See,e.g., International Patent Application Publication No. WO 00/43781). TheGDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer,inactivating its biological activity. See, e.g., Miyazono et al. (1988)J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol. Chem.,263; 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43. Otherproteins which bind to GDF8 or structurally related proteins and inhibittheir biological activity include follistatin, and potentially,follistatin-related proteins. See, e.g., Gamer et al. (1999) Dev. Biol.,208: 222-232.

GDF11, also known as BMP11, is a secreted protein that is expressed inthe tail bud, limb bud, maxillary and mandibular arches, and dorsal rootganglia during mouse development. See, e.g., McPherron et al. (1999)Nat. Genet., 22: 260-264; and Nakashima et al. (1999) Mech. Dev., 80:185-189. GDF11 plays a unique role in patterning both mesodermal andneural tissues. See, e.g., Gamer et al. (1999) Dev Biol., 208:222-32.GDF11 was shown to be a negative regulator of chondrogenesis andmyogenesis in developing chick limb. See, e.g., Gamer et al. (2001) DevBiol., 229:407-20. The expression of GDF11 in muscle also suggests itsrole in regulating muscle growth in a similar way to GDF8. In addition,the expression of GDF11 in brain suggests that GDF11 may also possessactivities that relate to the function of the nervous system.Interestingly, GDF11 was found to inhibit neurogenesis in the olfactoryepithelium. See, e.g., Wu et al. (2003) Neuron., 37:197-207. Hence,GDF11 may have in vitro and in vivo applications in the treatment ofdiseases such as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

BMP7, also called osteogenic protein-1 (OP-1), is well known to inducecartilage and bone formation. In addition, BMP7 regulates a wide arrayof physiological processes. For example, BMP7 may be the osteoinductivefactor responsible for the phenomenon of epithelial osteogenesis. It isalso found that BMP7 plays a role in calcium regulation and bonehomeostasis. Like activin, BMP7 binds to type II receptors, ActRIIA andActRIIB However, BMP7 and activin recruit distinct type I receptors intoheteromeric receptor complexes. The major BMP7 type I receptor observedwas ALK2, while activin bound exclusively to ALK4 (ActRIIB) BMP7 andactivin elicited distinct biological responses and activated differentSMAD pathways. See, e.g., Macias-Silva et al. (1998) J Biol Chem.273:25628-36.

Anti-Mullerian hormone (AMH), also known as Mullerian-inhibitingsubstance (MIS), is a TGF-beta family glycoprotein. One AMH-associatedtype II receptor has been identified and is designated as AMHRII, oralternatively MISRII. AMH induces regression of the Mullerian ducts inthe human male embryo. AMH is expressed in reproductive age women anddoes not fluctuate with cycle or pregnancy, but was found to graduallydecrease as both oocyte quantity and quality decrease, suggesting AMHcould serve as a biomarker for ovarian physiology. See e.g. Zec et al.,(2011) Biochemia Medica 21(3): 219-30.

Activin receptor-like kinase-1 (ALK1), the product of the ACVRL1 geneknown alternatively as ACVRLK1, is a type I receptor whose expression ispredominantly restricted to endothelial cells. See, e.g., OMIM entry601284. ALK1 is activated by the binding of TGF-beta family ligands suchas BMP9 and BMP10, and ALK1 signaling is critical in the regulation ofboth developmental and pathological blood vessel formation. ALK1expression overlaps with sites of vasculogenesis and angiogenesis inearly mouse development, and ALK1 knockout mice die around embryonic day11.5 because of severe vascular abnormalities (see e.g., Cunha andPietras (2011) Blood 117(26):6999-7006.) ALK1 expression has also beendescribed in other cell types such as hepatic stellate cells andchondrocytes. Additionally, ALK1 along with activin receptor-likekinase-2 (ALK2) have been found to be important for BMP9-inducedosteogenic signaling in mesenchymal stem cells. See e.g., Cunha andPietras (2011) Blood 117(26):6999-7006.

ALK2, the product of the ACVR1 gene known alternatively as ActRIA orACVRLK2, is a type I receptor that has been shown to bind activins andBMPs. ALK2 is critical for embryogenesis as ALK2 knockout mice die soonafter gastrulation. See, e.g., Mishina et al. (1999) Dev Biol. 213:314-326 and OMIM entry 102576. Constitutively active mutations in ALK2are associated with fibrodysplasia ossificans progressiva (FOP), a raregenetic disorder that causes fibrous tissue, including muscle, tendonand ligament, to be ossified spontaneously or when damaged. Anarginine-to-histidine mutation in position 206 of ALK2 is a naturallyoccurring mutation associated with FOP in humans. This mutation inducesBMP-specific signaling via ALK2 without the binding of ligand. See,e.g., Fukuda et al., (2009) J Biol Chem. 284(11):7149-7156 and Kaplan etal., (2011) Ann N.Y. Acad Sci. 1237: 5-10.

Activin receptor-like kinase-3 (ALK3), the product of the BMPR1A geneknown alternatively as ACVRLK3, is a type I receptor mediating effectsof multiple ligands in the BMP family. Unlike several type I receptorswith ubiquitous tissue expression, ALK3 displays a restricted pattern ofexpression consistent with more specialized functionality. See, e.g.,ten Dijke (1993) Oncogene, 8: 2879-2887 and OMIM entry 601299. ALK3 isgenerally recognized as a high-affinity receptor for BMP2, BMP4, BMP7and other members of the BMP family. BMP2 and BMP7 are potentstimulators of osteoblastic differentiation, and are now used clinicallyto induce bone formation in spine fusions and certain non-unionfractures. ALK3 is regarded as a key receptor in mediating BMP2 and BMP4signaling in osteoblasts. See, e.g., Lavery et al. (2008) J. Biol. Chem.283: 20948-20958. A homozygous ALK3 knockout mouse dies early inembryogenesis (˜day 9.5), however, adult mice carrying a conditionaldisruption of ALK3 in osteoblasts have been recently reported to exhibitincreased bone mass, although the newly formed bone showed evidence ofdisorganization. See, e.g., Kamiya (2008) J. Bone Miner. Res.,23:2007-2017; and Kamiya (2008) Development 135: 3801-3811. This findingis in startling contrast to the effectiveness of BMP2 and BMP7 (ligandsfor ALK3) as bone building agents in clinical use.

Activin receptor-like kinase-4 (ALK4), the product of the ACVR1B genealternatively known as ACVRLK4, is a type I receptor that transducessignaling for a number of TGF-beta family ligands including activins,nodal and GDFs. ALK4 mutations are associated with pancreatic cancer,and expression of dominant negative truncated ALK4 isoforms are highlyexpressed in human pituitary tumors. See, e.g., Tsuchida et al., (2008)Endocrine Journal 55(1):11-21 and OMIM entry 601300.

Activin receptor-like kinase-5 (ALK5), the product of the TGFBR1 gene,is widely expressed in most cell types. Several TGF-beta superfamilyligands, including TGF-betas, activin, and GDF-8, signal via ALK5 andactivate downstream Smad 2 and Smad 3. Mice deficient in ALK5 exhibitsevere defects in the vascular development of the yolk sac and placenta,lack circulating red blood cells, and die mid-gestation. It was foundthat these embryos had normal hematopoietic potential, but enhancedproliferation and improper migration of endothelial cells. Thus,ALK5-dependent signaling is important for angiogenesis, but not for thedevelopment of hematopoietic progenitor cells and functionalhematopoiesis. See, e.g. Larsson et al., (2001) The EMBO Journal, 20(7):1663-1673 and OMIM entry 190181. In endothelial cells, ALK5 actscooperatively and opposite to ALK1 signaling. ALK5 inhibits cellmigration and proliferation, notably the opposite effect of ALK1. See,e.g., Goumans et al. (2003) Mol Cell 12(4): 817-828. Additionally, ALK5is believed to negatively regulate muscle growth. Knockdown of ALK5 inthe muscle a mouse model of muscular dystrophy was found to decreasefibrosis and increase expression of genes associate with muscle growth.See, e.g. Kemaladewi et al., (2014) Mol Ther Nucleic Acids 3, e156.

Activin receptor-like kinase-6 (ALK6) is the product of the BMPR1B gene,whose deficiency is associated with chrondodysplasia and limb defects inboth humans and mice. See, e.g., Demirhan et al., (2005) J Med Genet.42:314-317. ALK6 is widely expressed throughout the developing skeleton,and is required for chondrogenesis in mice. See, e.g., Yi et al., (2000)Development 127:621-630 and OMIM entry 603248.

Activin receptor-like kinase-7 (ALK7) is the product of the ACVR1C gene.ALK7 null mice are viable, fertile, and display no skeletal or limbmalformations. GDF3 signaling through ALK7 appears to play a role ininsulin sensitivity and obesity. This is supported by results that ALK7null mice show reduced fat accumulation and resistance to diet-inducedobesity. See, e.g., Andersson et al., (2008) PNAS 105(20): 7252-7256.ALK7-mediated Nodal signaling has been implicated to have both tumorpromoting and tumor suppressing effects in a variety of different cancercell lines. See, e.g., De Silva et al., (2012) Frontiers inEndocrinology 3:59 and OMIM entry 608981.

As used herein the term “ActRII” refers to the family of type II activinreceptors. This family includes both the activin receptor type IIA(ActRIIA), encoded by the ACVR2A gene, and the activin receptor type IIB(ActRIIB), encoded by the ACVR2B gene. ActRII receptors are TGF-betasuperfamily type II receptors that bind a variety of TGF-betasuperfamily ligands including activins, GDF8 (myostatin), GDF11, and asubset of BMPs, notably BMP6 and BMP7. ActRII receptors are implicatedin a variety of biological disorders including muscle and neuromusculardisorders (e.g., muscular dystrophy, amyotrophic lateral sclerosis(ALS), and muscle atrophy), undesired bone/cartilage growth, adiposetissue disorders (e.g., obesity), metabolic disorders (e.g., type 2diabetes), and neurodegenerative disorders. See, e.g., Tsuchida et al.,(2008) Endocrine Journal 55(1):11-21, Knopf et al., U.S. Pat. No.8,252,900, and OMIM entries 102581 and 602730.

Transforming growth factor beta receptor II (TGFBRII), encoded by theTGFBR2 gene, is a type II receptor that is known to bind TGF-betaligands and activate downstream Smad 2 and Smad 3 effectors. See, e.g.,Hinck (2012) FEBS Letters 586: 1860-1870 and OMIM entry 190182. TGF-betasignaling through TGFBRII is critical in T-cell proliferation,maintenance of T regulatory cells and proliferation of precartilaginousstem cells. See, e.g., Li et al., (2006) Immunity 25(3): 455-471 andCheng et al., Int. J. Mol. Sci. 2014, 15, 12665-12676.

Bone morphogenetic protein receptor II (BMPRII), encoded by the BMPR2gene, is a type II receptor that is known to bind BMP ligands includingBMP7 and BMP4. Efficient ligand binding to BMPRII is dependent on thepresence of the appropriate TGFBR type I receptors. See, e.g.,Rosenzweig et al., (1995) PNAS 92:7632-7636. Mutations in BMPRII areassociated with pulmonary hypertension in humans. See OMIM entry 600799.

Müllerian-inhibiting substance receptor II (MISRII), the product of theAMHR2 gene known alternatively as anti-Müllerian hormone type IIreceptor, is a type II TGF-beta superfamily receptor. MISRII binds theMIS ligand, but requires the presence of an appropriate type I receptor,such as ALK3 or ALK6, for signal transduction. See, e.g., Hinck (2012)FEBS Letters 586:1860-1870 and OMIM entry 600956. MISRII is involved insex differentiation in humans and is required for Müllerian regressionin the human male. AMH is expressed in reproductive-age women and doesnot fluctuate with cycle or pregnancy, but was found to gradual decreaseas both oocyte quantity and quality decrease, suggesting AMH could serveas a biomarker of ovarian physiology. See, e.g., Zec et al., (2011)Biochemia Medica 21(3): 219-30 and OMIM entry 600956.

In certain aspects, the present disclosure relates to the use ofsingle-arm heteromultimer complexes comprising an extracellular domainof a TGFβ superfamily type I receptor polypeptide (e.g., ALK1, ALK2,ALK3, ALK4, ALK5, ALK6, and ALK7) or an extracellular domain of a TGFβsuperfamily type II receptor polypeptide (e.g., ActRIIA, ActRIIB,TGFBRII, BMPRII, and MISRII), preferably soluble heteromultimercomplexes, to antagonize intracellular signaling transduction (e.g.,Smad 2/3 and/or Smad 1/5/8 signaling) initiated by one or more TGFβsuperfamily ligands (e.g., activin A, activin B, activin C, activin E,activin AB, activin AC, activin AE, activin BC, activin BE, Nodal, GDF8,GDF11, BMP6 and/or BMP7). As described herein, such antagonistsingle-arm heteromultimer complexes may be useful for the treatment orprevention of various TGF-beta associated conditions, including withoutlimitation diseases and disorders associated with, for example, cancer,muscle, bone, fat, red blood cells, metabolism, fibrosis and othertissues that are affected by one or more ligands of the TGF-betasuperfamily.

In particular, the data of the present disclosure demonstrates thatsingle-arm heteromultimer complexes comprising an extracellular domainof a TGFβ superfamily type I receptor polypeptide or an extracellulardomain of a TGFβ superfamily type II receptor polypeptide have differentligand selectivity profiles in comparison to their correspondinghomomultimer complexes.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which it isused.

The terms “heteromer” or “heteromultimer” is a complex comprising atleast a first polypeptide and a second polypeptide, wherein the secondpolypeptide differs in amino acid sequence from the first polypeptide byat least one amino acid residue. The heteromer can comprise a“heterodimer” formed by the first and second polypeptide or can formhigher order structures where polypeptides in addition to the first andsecond polypeptide are present. Exemplary structures for theheteromultimer include heterodimers, heterotrimers, heterotetramers andfurther oligomeric structures. Heterodimers are designated herein as X:Yor equivalently as X—Y, where X represents a first polypeptide chain andY represents a second polypeptide chain. Higher-order heteromers andoligomeric structures are designated herein in a corresponding manner.In certain embodiments a heteromultimer is recombinant (e.g., one ormore polypeptide component may be a recombinant protein), isolatedand/or purified.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. However, in common usage andin the instant application, the term “homologous,” when modified with anadverb such as “highly,” may refer to sequence similarity and may or maynot relate to a common evolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

As used herein “does not substantially bind to X” is intended to meanthat an agent has a K_(D) that is greater than about 10⁻⁷, 10⁻⁶, 10⁻⁵,10⁻⁴ or greater (e.g., no detectable binding by the assay used todetermine the K_(D)) for “X”.

2. Heteromultimer Complexes Comprising Single-Arm TGFβ SuperfamilyReceptor Polypeptides

In certain aspects, the disclosure concerns heteromultimer proteincomplexes comprising one or more single-arm TGF-beta superfamily type Ior type II receptor polypeptides. In certain embodiments, thepolypeptides disclosed herein may form protein complexes comprising afirst polypeptide covalently or non-covalently associated with a secondpolypeptide, wherein the first polypeptide comprises the amino acidsequence of a type I or type II receptor polypeptide and the amino acidsequence of a first member of an interaction pair; and the secondpolypeptide comprises the amino acid sequence of a second member of theinteraction pair, and wherein the second polypeptide does not comprise atype I or type II receptor polypeptide. The interaction pair may be anytwo polypeptide sequences that interact to form a complex, particularlya heterodimeric complex although operative embodiments may also employan interaction pair that forms a homodimeric sequence. As describedherein, one member of the interaction pair may be fused to a type I ortype II receptor polypeptide, such as a polypeptide comprising an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10,11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46,47, 50, 51, 67, 68, 71, 72, 75, 76, 79, 80, 83, 84, 87, 88, 91, 92, 301,302, 305, 306, 309, 310, and 313. Preferably, the interaction pair isselected to confer an improved serum half-life, or to act as an adapteron to which another moiety, such as a polyethylene glycol moiety, isattached to provide an improved serum half-life relative to themonomeric form of the type I or type II receptor polypeptide.

As shown herein, monomeric (single-arm) forms of TGF-beta superfamilytype I or type II receptors can exhibit substantially alteredligand-binding selectivity compared to their corresponding homodimericforms, but the monomeric forms tend to have a short serum residence time(half-life), which is undesirable in the therapeutic setting. A commonmechanism for improving serum half-life is to express a polypeptide as ahomodimeric fusion protein with a constant domain portion (e.g., an Fcportion) of an IgG. However, TGF-beta superfamily receptor polypeptidesexpressed as homodimeric proteins (e.g., in an Fc fusion construct) maynot exhibit the same activity profile as the monomeric form. Asdemonstrated herein, the problem may be solved by fusing the monomericform to a half-life extending moiety, and surprisingly, this can bereadily achieved by expressing such proteins as an asymmetricheterodimeric fusion protein in which one member of an interaction pairis fused to a TGF-beta superfamily receptor polypeptide and anothermember of the interaction pair is fused to either no moiety or to aheterologous moiety, resulting in a novel ligand-binding profile coupledwith an improvement in serum half-life conferred by the interactionpair.

In certain aspects, the present disclosure relates to single-armheteromultimer complexes comprising at least one TGF-beta superfamilytype I receptor polypeptide (e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6,and ALK7 as well as SEQ ID NOs: 14, 15, 18, 19, 22, 23, 26, 27, 30, 31,34, 35, 38, 39, 83, 84, 87, 88, 91, 92, 301, 302, 305, 306, 309, 310,313) or at least one TGF-beta superfamily type II receptor polypeptide(e.g., ActRIIA, ActRIIB, TGFBRII, BMPRII, and MISRII as well SEQ ID NOs:1, 2, 3, 4, 5, 6, 9, 10, 11, 42, 43, 46, 47, 50, 51, 67, 68, 71, 72, 75,76, 79, and 80), which are generally referred to herein as “single-armheteromultimer complexes of the disclosure” or “TGF-beta superfamilyreceptor single-arm heteromultimer complexes”. Preferably, single-armheteromultimer complexes of the disclosure are soluble, e.g., asingle-arm heteromultimer complex comprises a soluble portion of atleast one TGFβ superfamily type I receptor polypeptide or a solubleportion of at least one TGFβ superfamily type II receptor polypeptide.In general, the extracellular domains of TGFβ superfamily type I andtype II receptors correspond to a soluble portion of the type I or typeII receptor. Therefore, in some embodiments, single-arm heteromultimercomplexes of the disclosure comprise an extracellular domain of a TGFβsuperfamily type I receptor polypeptide (e.g., one or more ALK1, ALK2,ALK3, ALK4, ALK5, ALK6, and/or ALK7 receptor extracellular domains) oran extracellular domain of a TGFβ superfamily type II receptorpolypeptide (e.g., one or more ActRIIA, ActRIIB, TGFBRII, BMPRII, and/orMISRII receptor extracellular domains). Exemplary extracellular domainsof ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ActRIIA, ActRIIB, TGFBRII,BMPRII, and MISRII are disclosed herein and such sequences, as well asfragments, functional variants, and modified forms thereof, may be usedin accordance with the inventions of the present disclosure (e.g.,single-arm heteromultimer complexes compositions and uses thereof).

A defining structural motif known as a three-finger toxin fold isimportant for ligand binding by type I and type II receptors and isformed by 10, 12, or 14 conserved cysteine residues located at varyingpositions within the extracellular domain of each monomeric receptor.See, e.g., Greenwald et al. (1999) Nat Struct Biol 6:18-22; Hinck (2012)FEBS Lett 586:1860-1870. Any of the heteromeric complexes describedherein may comprise such domain of a type I or type II receptor of theTGF-beta superfamily. The core ligand-binding domains of TGFβsuperfamily receptors, as demarcated by the outermost of these conservedcysteines, correspond to positions 29-109 of SEQ ID NO: 1 (ActRIIBprecursor); positions 30-110 of SEQ ID NO: 9 (ActRIIA precursor);positions 34-95 of SEQ ID NO: 14 (ALK1 precursor); positions 35-99 ofSEQ ID NO: 18 (ALK2 precursor); positions 61-130 of SEQ ID NO: 22 (ALK3precursor); positions 34-101 of SEQ ID NOs: 26 and 83 (ALK4 precursors);positions 36-106 of SEQ ID NOs: 30 and 87 (ALK 5 precursors); positions32-102 of SEQ ID NO: 34 (ALK6 isoform B precursor); positions 28-92 ofSEQ ID NOs: 38, 305, and 309 (ALK7 precursors); positions 51-143 of SEQID NO: 42 (TGFBRII isoform B precursor); positions 34-123 of SEQ ID NO:46 and 71 (BMPRII precursors); positions 24-116 of SEQ ID NO: 50, 75,and 79 (MISRII precursors); positions 44-168 of SEQ ID NO: 67 (TGFBRIIisoform A precursor); and positions 62-132 of SEQ ID NO: 91 (ALK6isoform A precursor). The structurally less-ordered amino acids flankingthese cysteine-demarcated core sequences can be truncated by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 residues oneither terminus without necessarily altering ligand binding. Exemplaryextracellular domains for N-terminal and/or C-terminal truncationinclude SEQ ID NOs: 2, 3, 5, 6, 10, 11 15, 19, 23, 27, 31, 35, 39, 43,47, 51, 68, 72, 76, 80, 84, 88, 92, 302, 306, 310, and 313.

In other preferred embodiments, single-arm heteromultimer complexes ofthe disclosure bind to and inhibit (antagonize) activity of one or moreTGF-beta superfamily ligands including, but not limited to, BMP2,BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10,GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11,GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C,activin E, activin AB, activin AC, activin AE, activin BC, activin BE,nodal, glial cell-derived neurotrophic factor (GDNF), neurturin,artemin, persephin, MIS, and Lefty. In particular, single-armheteromultimer complexes of the disclosure may be used to antagonizeintracellular signaling transduction (e.g., Smad 2/3 and/or Smad 1/5/8signaling) initiated by one or more TGFβ superfamily ligands. Asdescribed herein, such antagonist heteromultimer complexes may be forthe treatment or prevention of various TGF-beta associated conditions,including without limitation diseases and disorders associated with, forexample, cancer, muscle, bone, fat, red blood cells, metabolism,fibrosis and other tissues that are affected by one or more ligands ofthe TGF-beta superfamily. In some embodiments, single-arm heteromultimercomplexes of the disclosure have different ligand-binding profiles incomparison to their corresponding homomultimer complex (e.g., anActRIIB-Fc:Fc heterodimer vs. a corresponding ActRIIB-Fc:ActRIIB-Fc orFc:Fc homodimer). As described herein, single-arm heteromultimercomplexes of the disclosure include, e.g., heterodimers, heterotrimers,heterotetramers and further oligomeric structures based on a single-armunitary complex. In certain preferred embodiments, single-armheteromultimer complexes of the disclosure are heterodimers.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIB polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNo. WO 2006/012627, which is incorporated herein by reference in itsentirety. Numbering of amino acids for all ActRIIB-related polypeptidesdescribed herein is based on the numbering of the human ActRIIBprecursor protein sequence provided below (SEQ ID NO: 1), unlessspecifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER TNQSGLERCE51 GEQDKRLHCY ASWRNSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101 FCCCEGNFCNERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151 LIVLLAFWMY RHRKPPYGHVDIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSEREIFSTPGMK HENLLQFIAA 251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHVAETMSRGLSY 301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451 AQLCVTIEECWDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated with a single underline; theextracellular domain is indicated in bold font; and the potential,endogenous N-linked glycosylation sites are indicated with a doubleunderline.

The processed extracellular ActRIIB polypeptide sequence is as follows:

(SEQ ID NO: 2) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT.

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by a single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNE RFTHLPEA.

A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64)is also reported in the literature See, e.g., Hilden et al. (1994)Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fcfusion protein comprising an extracellular domain of ActRIIB with theA64 substitution has a relatively low affinity for activin and GDF11. Bycontrast, the same ActRIIB-Fc fusion protein with an arginine atposition 64 (R64) has an affinity for activin and GDF11 in the lownanomolar to high picomolar range. Therefore, sequences with an R64 areused as the “wild-type” reference sequence for human ActRIIB in thisdisclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 4) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER TNQSGLERCE51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101 FCCCEGNFCNERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151 LIVLLAFWMY RHRKPPYGHVDIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSEREIFSTPGMK HENLLQFIAA 251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHVAETMSRGLSY 301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451 AQLCVTIEECWDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated by single underline and theextracellular domain is indicated by bold font.

The processed extracellular ActRIIB polypeptide sequence of thealternative A64 form is as follows:

(SEQ ID NO: 5) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 6) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

A nucleic acid sequence encoding the human ActRIIB precursor protein isshown below (SEQ ID NO: 7), consisting of nucleotides 25-1560 of GenbankReference Sequence NM_001106.3, which encode amino acids 1-513 of theActRIIB precursor. The sequence as shown provides an arginine atposition 64 and may be modified to provide an alanine instead. Thesignal sequence is underlined.

(SEQ ID NO: 7) 1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG 101 CCAACTGGGAGCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA 151 GGCGAGCAGG ACAAGCGGCTGCACTGCTAC GCCTCCTGGC GCAACAGCTC 201 TGGCACCATC GAGCTCGTGA AGAAGGGCTGCTGGCTAGAT GACTTCAACT 251 GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCCCCAGGTGTAC 301 TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC351 AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA 401CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC 451 CTCATCGTCCTGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA 501 CGGTCATGTG GACATCCATGAGGACCCTGG GCCTCCACCA CCATCCCCTC 551 TGGTGGGCCT GAAGCCACTG CAGCTGCTGGAGATCAAGGC TCGGGGGCGC 601 TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTGTAGCTGTCAA 651 GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT701 TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC 751GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT 801 CCATGACAAGGGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT 851 GGAACGAACT GTGTCATGTAGCAGAGACGA TGTCACGAGG CCTCTCATAC 901 CTGCATGAGG ATGTGCCCTG GTGCCGTGGCGAGGGCCACA AGCCGTCTAT 951 TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAGAGCGACCTCA 1001 CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA1051 CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC 1101TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA 1151 TTGACATGTATGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC 1201 AAGGCTGCAG ACGGACCCGTGGATGAGTAC ATGCTGCCCT TTGAGGAAGA 1251 GATTGGCCAG CACCCTTCGT TGGAGGAGCTGCAGGAGGTG GTGGTGCACA 1301 AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACACCCGGGCCTG 1351 GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC1401 TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT 1451CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC 1501 ACCAATGTGGACCTGCCCCC TAAAGAGTCA AGCATC

A nucleic acid sequence encoding processed extracellular human ActRIIBpolypeptide is as follows (SEQ ID NO: 8). The sequence as shown providesan arginine at position 64, and may be modified to provide an alanineinstead.

(SEQ ID NO: 8) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC 101 AGGACAAGCGGCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC 151 ATCGAGCTCG TGAAGAAGGGCTGCTGGCTA GATGACTTCA ACTGCTACGA 201 TAGGCAGGAG TGTGTGGCCA CTGAGGAGAACCCCCAGGTG TACTTCTGCT 251 GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTTGCCAGAGGCT 301 GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC

An alignment of the amino acid sequences of human ActRIIB solubleextracellular domain and human ActRIIA soluble extracellular domain areillustrated in FIG. 3. This alignment indicates amino acid residueswithin both receptors that are believed to directly contact ActRIIligands. FIG. 4 depicts a multiple-sequence alignment of variousvertebrate ActRIIB proteins and human ActRIIA. From these alignments isit possible to predict key amino acid positions within theligand-binding domain that are important for normal ActRII-ligandbinding activities as well as to predict amino acid positions that arelikely to be tolerant to substitution without significantly alteringnormal ActRII-ligand binding activities. ActRII proteins have beencharacterized in the art in terms of structural and functionalcharacteristics, particularly with respect to ligand binding. See, e.g.,Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) NatureStructural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20:7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; aswell as U.S. Pat. Nos. 7,709,605, 7,612,041, and 7,842,663.

For example, Attisano et al. showed that a deletion of the proline knotat the C-terminus of the extracellular domain of ActRIIB reduced theaffinity of the receptor for activin. An ActRIIB-Fc fusion proteincontaining amino acids 20-119 of present SEQ ID NO: 1,“ActRIIB(20-119)-Fc”, has reduced binding to GDF11 and activin relativeto an ActRIIB(20-134)-Fc, which includes the proline knot region and thecomplete juxtamembrane domain (see, e.g., U.S. Pat. No. 7,842,663).However, an ActRIIB(20-129)-Fc protein retains similar but somewhatreduced activity relative to the wild-type, even though the proline knotregion is disrupted. Thus, ActRIIB extracellular domains that stop atamino acid 134, 133, 132, 131, 130 and 129 (with respect to SEQ IDNO: 1) are all expected to be active, but constructs stopping at 134 or133 may be most active. Similarly, mutations at any of residues 129-134(with respect to SEQ ID NO: 1) are not expected to alter ligand-bindingaffinity by large margins. In support of this, it is known in the artthat mutations of P129 and P130 (with respect to SEQ ID NO: 1) do notsubstantially decrease ligand binding. Therefore, an ActRIIB polypeptideof the present disclosure may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 (e.g., 109,110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected tohave reduced ligand binding. Amino acid 119 (with respect to present SEQID NO:1) is poorly conserved and so is readily altered or truncated.ActRIIB polypeptides and ActRIIB-based GDF traps ending at 128 (withrespect to SEQ ID NO: 1) or later should retain ligand-binding activity.ActRIIB polypeptides and ActRIIB-based GDF traps ending at or between119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127), withrespect to SEQ ID NO: 1, will have an intermediate binding ability. Anyof these forms may be desirable to use, depending on the clinical orexperimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before (with respect to SEQ ID NO: 1) will retainligand-binding activity. Amino acid 29 represents the initial cysteine.An alanine-to-asparagine mutation at position 24 (with respect to SEQ IDNO: 1) introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. See, e.g., U.S. Pat. No.7,842,663. This confirms that mutations in the region between the signalcleavage peptide and the cysteine cross-linked region, corresponding toamino acids 20-29, are well tolerated. In particular, ActRIIBpolypeptides and ActRIIB-based GDF traps beginning at position 20, 21,22, 23, and 24 (with respect to SEQ ID NO: 1) should retain generalligand-biding activity, and ActRIIB polypeptides and ActRIIB-based GDFtraps beginning at positions 25, 26, 27, 28, and 29 (with respect to SEQID NO: 1) are also expected to retain ligand-biding activity. Data shownin, e.g., U.S. Pat. No. 7,842,663 demonstrates that, surprisingly, anActRIIB construct beginning at 22, 23, 24, or 25 will have the mostactivity.

Taken together, an active portion (e.g., ligand-binding portion) ofActRIIB comprises amino acids 29-109 of SEQ ID NO: 1. Therefore ActRIIBpolypeptides of the present disclosure may, for example, comprise anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a portion of ActRIIB beginning at a residuecorresponding to amino acids 20-29 (e.g., beginning at amino acid 20,21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at aposition corresponding to amino acids 109-134 (e.g., ending at aminoacid 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) ofSEQ ID NO: 1. Other examples include polypeptides that begin at aposition from 20-29 (e.g., position 20, 21, 22, 23, 24, 25, 26, 27, 28,or 29) or 21-29 (e.g., position 21, 22, 23, 24, 25, 26, 27, 28, or 29)and end at a position from 119-134 (e.g., 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-133 (e.g.,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or133), 129-134 (e.g., 129, 130, 131, 132, 133, or 134), or 129-133 (e.g.,129, 130, 131, 132, or 133) of SEQ ID NO: 1. Other examples includeconstructs that begin at a position from 20-24 (e.g., 20, 21, 22, 23, or24), 21-24 (e.g., 21, 22, 23, or 24), or 22-25 (e.g., 22, 22, 23, or 25)and end at a position from 109-134 (e.g., 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134), 119-134 (e.g., 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) or129-134 (e.g., 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1.Variants within these ranges are also contemplated, particularly thosehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the corresponding portion of SEQ ID NO: 1.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 3, demonstrating that the ligand-bindingpocket is defined, in part, by residues Y31, N33, N35, L38 through T41,E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83,Y85, R87, A92, and E94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated. R40 is a K in Xenopus,indicating that basic amino acids at this position will be tolerated.Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, and therefore aminoacids including R, K, Q, N and H will be tolerated at this position.Thus, a general formula for an ActRIIB polypeptide of the disclosure isone that comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 ofSEQ ID NO: 1, optionally beginning at a position ranging from 20-24(e.g., 20, 21, 22, 23, or 24) or 22-25(e.g., 22, 23, 24, or 25) andending at a position ranging from 129-134 (e.g., 129, 130, 131, 132,133, or 134), and comprising no more than 1, 2, 5, 10 or 15 conservativeamino acid changes in the ligand-binding pocket, and zero, one or morenon-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82in the ligand-binding pocket. Sites outside the binding pocket, at whichvariability may be particularly well tolerated, include the amino andcarboxy termini of the extracellular domain (as noted above), andpositions 42-46 and 65-73 (with respect to SEQ ID NO: 1). Anasparagine-to-alanine alteration at position 65 (N65A) actually improvesligand binding in the A64 background, and is thus expected to have nodetrimental effect on ligand binding in the R64 background. See, e.g.,U.S. Pat. No. 7,842,663. This change probably eliminates glycosylationat N65 in the A64 background, thus demonstrating that a significantchange in this region is likely to be tolerated. While an R64A change ispoorly tolerated, R64K is well-tolerated, and thus another basicresidue, such as H may be tolerated at position 64. See, e.g., U.S. Pat.No. 7,842,663.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant polypeptide useful in accordance with thepresently disclosed methods may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Yin Xenopus, and therefore Y or other hydrophobicgroup, such as I, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas P in rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

The variations described herein may be combined in various ways.Additionally, the results of the mutagenesis program described in theart indicate that there are amino acid positions in ActRIIB that areoften beneficial to conserve. With respect to SEQ ID NO: 1, theseinclude position 64 (basic amino acid), position 80 (acidic orhydrophobic amino acid), position 78 (hydrophobic, and particularlytryptophan), position 37 (acidic, and particularly aspartic or glutamicacid), position 56 (basic amino acid), position 60 (hydrophobic aminoacid, particularly phenylalanine or tyrosine). Thus, in the ActRIIBpolypeptides disclosed herein, the disclosure provides a framework ofamino acids that may be conserved. Other positions that may be desirableto conserve are as follows: position 52 (acidic amino acid), position 55(basic amino acid), position 81 (acidic), 98 (polar or charged,particularly E, D, R or K), all with respect to SEQ ID NO: 1.

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ActRIIB polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ActRIIB polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ActRIIB polypeptide and uses thereof) are soluble (e.g.,an extracellular domain of ActRIIB) In other preferred embodiments,ActRIIB polypeptides for use in accordance with the inventions of thedisclosure bind to and/or inhibit (antagonize) activity (e.g., inductionof Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-betasuperfamily ligands. In some embodiments, single-arm heteromultimercomplexes of the disclosure comprise at least one ActRIIB polypeptidethat comprises, consists, or consists essentially of an amino acidsequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to a portion of ActRIIB beginning at a residuecorresponding to amino acids 20-29 (e.g., beginning at amino acid 20,21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at aposition corresponding to amino acids 109-134 (e.g., ending at aminoacid 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) ofSEQ ID NO: 1. In some embodiments, single-arm heteromultimer complexesof the disclosure comprise at least one ActRIIB polypeptide thatcomprises, consists, or consists essentially of an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a portion of ActRIIB beginning at a residue correspondingto amino acids 20-29 (e.g., beginning at amino acid 20, 21, 22, 23, 24,25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at a positioncorresponding to amino acids 109-134 (e.g., ending at amino acid 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO:1, wherein the position corresponding to L79 of SEQ ID NO: 1 is anacidic amino acid (i.e., a D or E amino acid residue). In certainpreferred embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ActRIIB polypeptide that comprises,consists, or consists essentially of an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical aminoacids 29-109 of SEQ ID NO: 1. In other preferred embodiments, single-armheteromultimer complexes of the disclosure comprise at least one ActRIIBpolypeptide that comprises, consists, or consists essentially of anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical amino acids 29-109 of SEQ ID NO: 1, wherein theposition corresponding to L79 of SEQ ID NO: 1 is an acidic amino acid(i.e., a D or E amino acid residue). In some embodiments, single-armheteromultimer complexes of the disclosure comprise at least one ActRIIBpolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2,3, 4, 5, 6, 104, 106, 403, or 404. In some embodiments, single-armheteromultimer complexes of the disclosure comprise at least one ActRIIBpolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2,3, 4, 5, 6, 104, 106, 403, or 404, wherein the position corresponding toL79 of SEQ ID NO: 1 is an acidic amino acid (i.e., a D or E amino acidresidue). In some embodiments, single-arm heteromultimer complexes ofthe disclosure comprise, consist, or consist essentially of at least oneActRIIB polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% identical to the amino acid sequence of any one of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 104, 106, 403, or 404. In some embodiments,single-arm heteromultimer complexes of the disclosure comprise, consist,or consist essentially of at least one ActRIIB polypeptide that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 104,106, 403, or 404, wherein the position corresponding to L79 of SEQ IDNO: 1 is an acidic amino acid (i.e., a D or E amino acid residue).

In certain embodiments, the present disclosure relates to a proteincomplex comprising an ActRIIA polypeptide. As used herein, the term“ActRIIA” refers to a family of activin receptor type IIA (ActRIIA)proteins from any species and variants derived from such ActRIIAproteins by mutagenesis or other modification. Reference to ActRIIAherein is understood to be a reference to any one of the currentlyidentified forms. Members of the ActRIIA family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain comprising a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The term “ActRIIA polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIA family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIA polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNo. WO 2006/012627, which is incorporated herein by reference in itsentirety. Numbering of amino acids for all ActRIIA-related polypeptidesdescribed herein is based on the numbering of the human ActRIIAprecursor protein sequence provided below (SEQ ID NO: 9), unlessspecifically designated otherwise.

The human ActRIIA precursor protein sequence is as follows:

(SEQ ID NO: 9) 1 MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RTNQTGVEPC51 YGDKDKRRHC FATWKNISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV 101 YFCCCEGNMCNEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI 151 AGIVICAFWV YRHHKMAYPPVLVPTQDPGP PPPSPLLGLK PLQLLEVKAR 201 GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQNEYEVYSLPG MKHENILQFI 251 GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELCHIAETMARGL 301 AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGLALKFEAG351 KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR 401CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG 451 MAMLCETIEECWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM 501 VTNVDFPPKE SSL

The signal peptide is indicated by a single underline; the extracellulardomain is indicated in bold font; and the potential, endogenous N-linkedglycosylation sites are indicated by a double underline.

The processed extracellular human ActRIIA polypeptide sequence is asfollows:

(SEQ ID NO: 10) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is indicated by asingle underline. The sequence with the “tail” deleted (a Δ15 sequence)is as follows:

(SEQ ID NO: 11) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM

A nucleic acid sequence encoding the human ActRIIA precursor protein isshown below (SEQ ID NO: 12), corresponding to nucleotides 159-1700 ofGenbank Reference Sequence NM_001616.4. The signal sequence isunderlined.

(SEQ ID NO: 12) 1 ATGGGAGCTG CTGCAAAGTT GGCGTTTGCC GTCTTTCTTA TCTCCTGTTC51 TTCAGGTGCT ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA 101 ATGCTAATTGGGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT 151 TATGGTGACA AAGATAAACGGCGGCATTGT TTTGCTACCT GGAAGAATAT 201 TTCTGGTTCC ATTGAAATAG TGAAACAAGGTTGTTGGCTG GATGATATCA 251 ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAGCCCTGAAGTA 301 TATTTTTGTT GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT351 TCCGGAGATG GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC 401CACCCTATTA CAACATCCTG CTCTATTCCT TGGTGCCACT TATGTTAATT 451 GCGGGGATTGTCATTTGTGC ATTTTGGGTG TACAGGCATC ACAAGATGGC 501 CTACCCTCCT GTACTTGTTCCAACTCAAGA CCCAGGACCA CCCCCACCTT 551 CTCCATTACT AGGTTTGAAA CCACTGCAGTTATTAGAAGT GAAAGCAAGG 601 GGAAGATTTG GTTGTGTCTG GAAAGCCCAG TTGCTTAACGAATATGTGGC 651 TGTCAAAATA TTTCCAATAC AGGACAAACA GTCATGGCAA AATGAATACG701 AAGTCTACAG TTTGCCTGGA ATGAAGCATG AGAACATATT ACAGTTCATT 751GGTGCAGAAA AACGAGGCAC CAGTGTTGAT GTGGATCTTT GGCTGATCAC 801 AGCATTTCATGAAAAGGGTT CACTATCAGA CTTTCTTAAG GCTAATGTGG 851 TCTCTTGGAA TGAACTGTGTCATATTGCAG AAACCATGGC TAGAGGATTG 901 GCATATTTAC ATGAGGATAT ACCTGGCCTAAAAGATGGCC ACAAACCTGC 951 CATATCTCAC AGGGACATCA AAAGTAAAAA TGTGCTGTTGAAAAACAACC 1001 TGACAGCTTG CATTGCTGAC TTTGGGTTGG CCTTAAAATT TGAGGCTGGC1051 AAGTCTGCAG GCGATACCCA TGGACAGGTT GGTACCCGGA GGTACATGGC 1101TCCAGAGGTA TTAGAGGGTG CTATAAACTT CCAAAGGGAT GCATTTTTGA 1151 GGATAGATATGTATGCCATG GGATTAGTCC TATGGGAACT GGCTTCTCGC 1201 TGTACTGCTG CAGATGGACCTGTAGATGAA TACATGTTGC CATTTGAGGA 1251 GGAAATTGGC CAGCATCCAT CTCTTGAAGACATGCAGGAA GTTGTTGTGC 1301 ATAAAAAAAA GAGGCCTGTT TTAAGAGATT ATTGGCAGAAACATGCTGGA 1351 ATGGCAATGC TCTGTGAAAC CATTGAAGAA TGTTGGGATC ACGACGCAGA1401 AGCCAGGTTA TCAGCTGGAT GTGTAGGTGA AAGAATTACC CAGATGCAGA 1451GACTAACAAA TATTATTACC ACAGAGGACA TTGTAACAGT GGTCACAATG 1501 GTGACAAATGTTGACTTTCC TCCCAAAGAA TCTAGTCTA

The nucleic acid sequence encoding processed extracellular ActRIIApolypeptide is as follows:

(SEQ ID NO: 13) 1 ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA ATGCTAATTG51 GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT TATGGTGACA 101 AAGATAAACGGCGGCATTGT TTTGCTACCT GGAAGAATAT TTCTGGTTCC 151 ATTGAAATAG TGAAACAAGGTTGTTGGCTG GATGATATCA ACTGCTATGA 201 CAGGACTGAT TGTGTAGAAA AAAAAGACAGCCCTGAAGTA TATTTTTGTT 251 GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTTTCCGGAGATG 301 GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC CACCC

A general formula for an active (e.g., ligand binding) ActRIIApolypeptide is one that comprises a polypeptide that starts at aminoacid 30 and ends at amino acid 110 of SEQ ID NO: 9. Accordingly, ActRIIApolypeptides of the present disclosure may comprise a polypeptide thatis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 30-110 of SEQ ID NO: 9. Optionally, ActRIIA polypeptides ofthe present disclosure comprise a polypeptide that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids aminoacids 12-82 of SEQ ID NO: 9 optionally beginning at a position rangingfrom 1-5 (e.g., 1, 2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) and endingat a position ranging from 110-116 (e.g., 110, 111, 112, 113, 114, 115,or 116) or 110-115 (e.g., 110, 111, 112, 113, 114, or 115),respectively, and comprising no more than 1, 2, 5, 10 or 15 conservativeamino acid changes in the ligand binding pocket, and zero, one or morenon-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82in the ligand-binding pocket with respect to SEQ ID NO: 9.

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ActRIIA polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ActRIIA polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ActRIIA polypeptide and uses thereof) are soluble (e.g.,an extracellular domain of ActRIIA). In other preferred embodiments,ActRIIA polypeptides for use in accordance with the inventions of thedisclosure bind to and/or inhibit (antagonize) activity (e.g., inductionof Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-betasuperfamily ligands. In some embodiments, single-arm heteromultimercomplexes of the disclosure comprise at least one ActRIIA polypeptidethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 9, 10,11, 101, 103, 401, or 402. In some embodiments, single-armheteromultimer complexes of the disclosure comprise, consist, or consistessentially of at least one ActRIIA polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of any one of SEQ ID NOs: 9, 10, 11, 101, 103, 401, or 402.

In certain aspects, the present disclosure relates to protein complexesthat comprise a TGFBRII polypeptide. As used herein, the term “TGFBRII”refers to a family of transforming growth factor-beta receptor II(TGFBRII) proteins from any species and variants derived from suchproteins by mutagenesis or other modification. Reference to TGFBRIIherein is understood to be a reference to any one of the currentlyidentified forms. Members of the TGFBRII family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain with a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The term “TGFBRII polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a TGFBRII family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all TGFBRII-related polypeptides described herein is based onthe numbering of the human TGFBRII precursor protein sequence below (SEQID NO: 42), unless specifically designated otherwise.

The canonical human TGFBRII precursor protein sequence (NCBI Ref SeqNP_003233.4) is as follows:

(SEQ ID NO: 42) 1 MGRGLLRGLW PLHIVLWTRI AS TIPPHVQK SVNNDMIVTDNNGAVKFPQL 51 CKFCDVRFST CDNQKSCMSN CSITSICEKP QEVCVAVWRK NDENITLETV 101CHDPKLPYHD FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFS 151 EEYNTSNPDLLLVIFQVTGI SLLPPLGVAI SVIIIFYCYR VNRQQKLSST 201 WETGKTRKLM EFSEHCAIILEDDRSDISST CANNINHNTE LLPIELDTLV 251 GKGRFAEVYK AKLKQNTSEQ FETVAVKIFPYEEYASWKTE KDIFSDINLK 301 HENILQFLTA EERKTELGKQ YWLITAFHAK GNLQEYLTRHVISWEDLRKL 351 GSSLARGIAH LHSDHTPCGR PKMPIVHRDL KSSNILVKND LTCCLCDFGL401 SLRLDPTLSV DDLANSGQVG TARYMAPEVL ESRMNLENVE SFKQTDVYSM 451ALVLWEMTSR CNAVGEVKDY EPPFGSKVRE HPCVESMKDN VLRDRGRPEI 501 PSFWLNHQGIQMVCETLTEC WDHDPEARLT AQCVAERFSE LEHLDRLSGR 551 SCSEEKIPED GSLNTTK

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular TGFBRII polypeptide sequence is as follows:

(SEQ ID NO: 43) TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ

The nucleic acid sequence encoding TGFBRII precursor protein is shownbelow (SEQ ID NO:44), corresponding to nucleotides 383-2083 of GenbankReference Sequence NM_003242.5. The signal sequence is underlined.

(SEQ ID NO: 44) ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGC ACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCAACCTGGGAAACCGGCAAGACGCGGAAGCTCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGATGACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATCAACCACAACACAGAGCTGCTGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTTGCTGAGGTCTATAAGGCCAAGCTGAAGCAGAACACTTCAGAGCAGTTTGAGACAGTGGCAGTCAAGATCTTTCCCTATGAGGAGTATGCCTCTTGGAAGACAGAGAAGGACATCTTCTCAGACATCAATCTGAAGCATGAGAACATACTCCAGTTCCTGACGGCTGAGGAGCGGAAGACGGAGTTGGGGAAACAATACTGGCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACGCGGCATGTCATCAGCTGGGAGGACCTGCGCAAGCTGGGCAGCTCCCTCGCCCGGGGGATTGCTCACCTCCACAGTGATCACACTCCATGTGGGAGGCCCAAGATGCCCATCGTGCACAGGGACCTCAAGAGCTCCAATATCCTCGTGAAGAACGACCTAACCTGCTGCCTGTGTGACTTTGGGCTTTCCCTGCGTCTGGACCCTACTCTGTCTGTGGATGACCTGGCTAACAGTGGGCAGGTGGGAACTGCAAGATACATGGCTCCAGAAGTCCTAGAATCCAGGATGAATTTGGAGAATGTTGAGTCCTTCAAGCAGACCGATGTCTACTCCATGGCTCTGGTGCTCTGGGAAATGACATCTCGCTGTAATGCAGTGGGAGAAGTAAAAGATTATGAGCCTCCATTTGGTTCCAAGGTGCGGGAGCACCCCTGTGTCGAAAGCATGAAGGACAACGTGTTGAGAGATCGAGGGCGACCAGAAATTCCCAGCTTCTGGCTCAACCACCAGGGCATCCAGATGGTGTGTGAGACGTTGACTGAGTGCTGGGACCACGACCCAGAGGCCCGTCTCACAGCCCAGTGTGTGGCAGAACGCTTCAGTGAGCTGGAGCATCTGGACAGGCTCTCGGGGAGGAGCTGCTCGGAGGAGAAGATTCCTGAAGACGGCTCCCTAAACACTACCAA A

The nucleic acid sequence encoding processed extracellular TGFBRIIpolypeptide is as follows:

(SEQ ID NO: 45) ACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAA

An alternative isoform of TGFBRII, isoform A (NP_001020018.1), is asfollows:

(SEQ ID NO: 67) 1 MGRGLLRGLW PLHIVLWTRI AS TIPPHVQK SDVEMEAQKDEIICPSCNRT 51 AHPLRHINND MIVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS 101ICEKPQEVCV AVWRKNDENI TLETVCHDPK LPYHDFILED AASPKCIMKE 151 KKKPGETFFMCSCSSDECND NIIFSEEYNT SNPDLLLVIF QVTGISLLPP 201 LGVAISVIII FYCYRVNRQQKLSSTWETGK TRKLMEFSEH CAIILEDDRS 251 DISSTCANNI NHNTELLPIE LDTLVGKGRFAEVYKAKLKQ NTSEQFETVA 301 VKIFPYEEYA SWKTEKDIFS DINLKHENIL QFLTAEERKTELGKQYWLIT 351 AFHAKGNLQE YLTRHVISWE DLRKLGSSLA RGIAHLHSDH TPCGRPKMPI401 VHRDLKSSNI LVKNDLTCCL CDFGLSLRLD PTLSVDDLAN SGQVGTARYM 451APEVLESRMN LENVESFKQT DVYSMALVLW EMTSRCNAVG EVKDYEPPFG 501 SKVREHPCVESMKDNVLRDR GRPEIPSFWL NHQGIQMVCE TLTECWDHDP 551 EARLTAQCVA ERFSELEHLDRLSGRSCSEE KIPEDGSLNT TK

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular TGFBRII polypeptide sequence (isoform A) isas follows:

(SEQ ID NO: 68) TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDLLLVIFQ

A nucleic acid sequence encoding the TGFBRII precursor protein (isoformA) is shown below (SEQ ID NO: 69), corresponding to nucleotides 383-2158of Genbank Reference Sequence NM_001024847.2. The signal sequence isunderlined.

(SEQ ID NO: 69) ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGC ACGATCCCACCGCACGTTCAGAAGTCGGATGTGGAAATGGAGGCCCAGAAAGATGAAATCATCTGCCCCAGCTGTAATAGGACTGCCCATCCACTGAGACATATTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCAACCTGGGAAACCGGCAAGACGCGGAAGCTCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGATGACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATCAACCACAACACAGAGCTGCTGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTTGCTGAGGTCTATAAGGCCAAGCTGAAGCAGAACACTTCAGAGCAGTTTGAGACAGTGGCAGTCAAGATCTTTCCCTATGAGGAGTATGCCTCTTGGAAGACAGAGAAGGACATCTTCTCAGACATCAATCTGAAGCATGAGAACATACTCCAGTTCCTGACGGCTGAGGAGCGGAAGACGGAGTTGGGGAAACAATACTGGCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACGCGGCATGTCATCAGCTGGGAGGACCTGCGCAAGCTGGGCAGCTCCCTCGCCCGGGGGATTGCTCACCTCCACAGTGATCACACTCCATGTGGGAGGCCCAAGATGCCCATCGTGCACAGGGACCTCAAGAGCTCCAATATCCTCGTGAAGAACGACCTAACCTGCTGCCTGTGTGACTTTGGGCTTTCCCTGCGTCTGGACCCTACTCTGTCTGTGGATGACCTGGCTAACAGTGGGCAGGTGGGAACTGCAAGATACATGGCTCCAGAAGTCCTAGAATCCAGGATGAATTTGGAGAATGTTGAGTCCTTCAAGCAGACCGATGTCTACTCCATGGCTCTGGTGCTCTGGGAAATGACATCTCGCTGTAATGCAGTGGGAGAAGTAAAAGATTATGAGCCTCCATTTGGTTCCAAGGTGCGGGAGCACCCCTGTGTCGAAAGCATGAAGGACAACGTGTTGAGAGATCGAGGGCGACCAGAAATTCCCAGCTTCTGGCTCAACCACCAGGGCATCCAGATGGTGTGTGAGACGTTGACTGAGTGCTGGGACCACGACCCAGAGGCCCGTCTCACAGCCCAGTGTGTGGCAGAACGCTTCAGTGAGCTGGAGCATCTGGACAGGCTCTCGGGGAGGAGCTGCTCGGAGGAGAAGATTCCTGAAGACGGCTCCCTAAACACTACCAAA

A nucleic acid sequence encoding the processed extracellular TGFBRIIpolypeptide (isoform A) is as follows:

(SEQ ID NO: 70) ACGATCCCACCGCACGTTCAGAAGTCGGATGTGGAAATGGAGGCCCAGAAAGATGAAATCATCTGCCCCAGCTGTAATAGGACTGCCCATCCACTGAGACATATTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCAT ATTTCAA.

Either of the foregoing TGFβRII isoforms (SEQ ID NOs: 42, 43, 67, and68) could incorporate an insertion of 36 amino acids (SEQ ID NO: 95)between the pair of glutamate residues (positions 151 and 152 of SEQ IDNO: 42; positions 129 and 130 of SEQ ID NO: 43; positions 176 and 177 ofSEQ ID NO: 67; or positions 154 and 155 of SEQ ID NO: 68) located nearthe C-terminus of the TGFβRII ECD, as occurs naturally in the TGFβRIIisoform C (Konrad et al., BMC Genomics 8:318, 2007).

(SEQ ID NO: 95) GRCKIRHIGS NNRLQRSTCQ NTGWESAHVM KTPGFR

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one TGFBRII polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, TGFBRII polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising a TGFBRII polypeptide and uses thereof) are soluble (e.g., anextracellular domain of TGFBRII). In other preferred embodiments,TGFBRII polypeptides for use in accordance with the inventions of thedisclosure bind to and/or inhibit (antagonize) activity (e.g., inductionof Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-betasuperfamily ligands. In some embodiments, single-arm heteromultimercomplexes of the disclosure comprise at least one TGFBRII polypeptidethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NOs: 42, 43, 67, 68, 113,115, 409, or 410. In some embodiments, single-arm heteromultimercomplexes of the disclosure comprise at least one TGFBRII polypeptidethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to any of the amino acid sequences of SEQ ID NOs: 42, 43, 67,68, 113, 115, 409, or 410, into which is inserted SEQ ID NO: 95 betweenthe paired glutamate residues as described above. In some embodiments,single-arm heteromultimer complexes of the disclosure consist or consistessentially of at least one TGFBRII polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NOs: 42, 43, 67, 68, 113, 115, 409, or 410.

In certain aspects, the present disclosure relates to protein complexesthat comprise a BMPRII polypeptide. As used herein, the term “BMPRII”refers to a family of bone morphogenetic protein receptor type II(BMPRII) proteins from any species and variants derived from such BMPRIIproteins by mutagenesis or other modification. Reference to BMPRIIherein is understood to be a reference to any one of the currentlyidentified forms. Members of the BMPRII family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain with a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The term “BMPRII polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a BMPRII family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all BMPRII-related polypeptides described herein is based onthe numbering of the human BMPRII precursor protein sequence below (SEQID NO: 46), unless specifically designated otherwise.

The canonical human BMPRII precursor protein sequence (NCBI Ref SeqNP_001195.2) is as follows:

(SEQ ID NO: 46) 1 MTSSLQRPWR VPWLPWTILL VSTAAA SQNQ ERLCAFKDPYQQDLGIGESR 51 ISHENGTILC SKGSTCYGLW EKSKGDINLV KQGCWSHIGD PQECHYEECV 101VTTTPPSIQN GTYRFCCCST DLCNVNFTEN FPPPDTTPLS PPHSFNRDET 151 IIIALASVSVLAVLIVALCF GYRMLTGDRK QGLHSMNMME AAASEPSLDL 201 DNLKLLELIG RGRYGAVYKGSLDERPVAVK VFSFANRQNF INEKNIYRVP 251 LMEHDNIARF IVGDERVTAD GRMEYLLVMEYYPNGSLCKY LSLHTSDWVS 301 SCRLAHSVTR GLAYLHTELP RGDHYKPAIS HRDLNSRNVLVKNDGTCVIS 351 DFGLSMRLTG NRLVRPGEED NAAISEVGTI RYMAPEVLEG AVNLRDCESA401 LKQVDMYALG LIYWEIFMRC TDLFPGESVP EYQMAFQTEV GNHPTFEDMQ 451VLVSREKQRP KFPEAWKENS LAVRSLKETI EDCWDQDAEA RLTAQCAEER 501 MAELMMIWERNKSVSPTVNP MSTAMQNERN LSHNRRVPKI GPYPDYSSSS 551 YIEDSIHHTD SIVKNISSEHSMSSTPLTIG EKNRNSINYE RQQAQARIPS 601 PETSVTSLST NTTTTNTTGL TPSTGMTTISEMPYPDETNL HTTNVAQSIG 651 PTPVCLQLTE EDLETNKLDP KEVDKNLKES SDENLMEHSLKQFSGPDPLS 701 STSSSLLYPL IKLAVEATGQ QDFTQTANGQ ACLIPDVLPT QIYPLPKQQN751 LPKRPTSLPL NTKNSTKEPR LKFGSKHKSN LKQVETGVAK MNTINAAEPH 801VVTVTMNGVA GRNHSVNSHA ATTQYANGTV LSGQTTNIVT HRAQEMLQNQ 851 FIGEDTRLNINSSPDEHEPL LRREQQAGHD EGVLDRLVDR RERPLEGGRT 901 NSNNNNSNPC SEQDVLAQGVPSTAADPGPS KPRRAQRPNS LDLSATNVLD 951 GSSIQIGEST QDGKSGSGEK IKKRVKTPYSLKRWRPSTWV ISTESLDCEV 1001 NNNGSNRAVH SKSSTAVYLA EGGTATTMVS KDIGMNCL

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular BMPRII polypeptide sequence is as follows:

(SEQ ID NO: 47) SQNQERLCAFKDPYQQDLGIGESRISHENGTILCSKGSTCYGLWEKSKGDINLVKQGCWSHIGDPQECHYEECVVTTTPPSIQNGTYRFCCCSTDLCNVNFTENFPPPDTTPLSPPHSFNRDET

A nucleic acid sequence encoding BMPRII precursor protein is shown below(SEQ ID NO: 48), as follows nucleotides 1149-4262 of Genbank ReferenceSequence NM_001204.6. The signal sequence is underlined.

(SEQ ID NO: 48) ATGACTTCCTCGCTGCAGCGGCCCTGGCGGGTGCCCTGGCTACCATGGACCATCCTGCTGGTCAGCACTGCGGCTGCTTCGCAGAATCAAGAACGGCTATGTGCGTTTAAAGATCCGTATCAGCAAGACCTTGGGATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCGAAAGGTAGCACCTGCTATGGCCTTTGGGAGAAATCAAAAGGGGACATAAATCTTGTAAAACAAGGATGTTGGTCTCACATTGGAGATCCCCAAGAGTGTCACTATGAAGAATGTGTAGTAACTACCACTCCTCCCTCAATTCAGAATGGAACATACCGTTTCTGCTGTTGTAGCACAGATTTATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGACACAACACCACTCAGTCCACCTCATTCATTTAACCGAGATGAGACAATAATCATTGCTTTGGCATCAGTCTCTGTATTAGCTGTTTTGATAGTTGCCTTATGCTTTGGATACAGAATGTTGACAGGAGACCGTAAACAAGGTCTTCACAGTATGAACATGATGGAGGCAGCAGCATCCGAACCCTCTCTTGATCTAGATAATCTGAAACTGTTGGAGCTGATTGGCCGAGGTCGATATGGAGCAGTATATAAAGGCTCCTTGGATGAGCGTCCAGTTGCTGTAAAAGTGTTTTCCTTTGCAAACCGTCAGAATTTTATCAACGAAAAGAACATTTACAGAGTGCCTTTGATGGAACATGACAACATTGCCCGCTTTATAGTTGGAGATGAGAGAGTCACTGCAGATGGACGCATGGAATATTTGCTTGTGATGGAGTACTATCCCAATGGATCTTTATGCAAGTATTTAAGTCTCCACACAAGTGACTGGGTAAGCTCTTGCCGTCTTGCTCATTCTGTTACTAGAGGACTGGCTTATCTTCACACAGAATTACCACGAGGAGATCATTATAAACCTGCAATTTCCCATCGAGATTTAAACAGCAGAAATGTCCTAGTGAAAAATGATGGAACCTGTGTTATTAGTGACTTTGGACTGTCCATGAGGCTGACTGGAAATAGACTGGTGCGCCCAGGGGAGGAAGATAATGCAGCCATAAGCGAGGTTGGCACTATCAGATATATGGCACCAGAAGTGCTAGAAGGAGCTGTGAACTTGAGGGACTGTGAATCAGCTTTGAAACAAGTAGACATGTATGCTCTTGGACTAATCTATTGGGAGATATTTATGAGATGTACAGACCTCTTCCCAGGGGAATCCGTACCAGAGTACCAGATGGCTTTTCAGACAGAGGTTGGAAACCATCCCACTTTTGAGGATATGCAGGTTCTCGTGTCTAGGGAAAAACAGAGACCCAAGTTCCCAGAAGCCTGGAAAGAAAATAGCCTGGCAGTGAGGTCACTCAAGGAGACAATCGAAGACTGTTGGGACCAGGATGCAGAGGCTCGGCTTACTGCACAGTGTGCTGAGGAAAGGATGGCTGAACTTATGATGATTTGGGAAAGAAACAAATCTGTGAGCCCAACAGTCAATCCAATGTCTACTGCTATGCAGAATGAACGCAACCTGTCACATAATAGGCGTGTGCCAAAAATTGGTCCTTATCCAGATTATTCTTCCTCCTCATACATTGAAGACTCTATCCATCATACTGACAGCATCGTGAAGAATATTTCCTCTGAGCATTCTATGTCCAGCACACCTTTGACTATAGGGGAAAAAAACCGAAATTCAATTAACTATGAACGACAGCAAGCACAAGCTCGAATCCCCAGCCCTGAAACAAGTGTCACCAGCCTCTCCACCAACACAACAACCACAAACACCACAGGACTCACGCCAAGTACTGGCATGACTACTATATCTGAGATGCCATACCCAGATGAAACAAATCTGCATACCACAAATGTTGCACAGTCAATTGGGCCAACCCCTGTCTGCTTACAGCTGACAGAAGAAGACTTGGAAACCAACAAGCTAGACCCAAAAGAAGTTGATAAGAACCTCAAGGAAAGCTCTGATGAGAATCTCATGGAGCACTCTCTTAAACAGTTCAGTGGCCCAGACCCACTGAGCAGTACTAGTTCTAGCTTGCTTTACCCACTCATAAAACTTGCAGTAGAAGCAACTGGACAGCAGGACTTCACACAGACTGCAAATGGCCAAGCATGTTTGATTCCTGATGTTCTGCCTACTCAGATCTATCCTCTCCCCAAGCAGCAGAACCTTCCCAAGAGACCTACTAGTTTGCCTTTGAACACCAAAAATTCAACAAAAGAGCCCCGGCTAAAATTTGGCAGCAAGCACAAATCAAACTTGAAACAAGTCGAAACTGGAGTTGCCAAGATGAATACAATCAATGCAGCAGAACCTCATGTGGTGACAGTCACCATGAATGGTGTGGCAGGTAGAAACCACAGTGTTAACTCCCATGCTGCCACAACCCAATATGCCAATGGGACAGTACTATCTGGCCAAACAACCAACATAGTGACACATAGGGCCCAAGAAATGTTGCAGAATCAGTTTATTGGTGAGGACACCCGGCTGAATATTAATTCCAGTCCTGATGAGCATGAGCCTTTACTGAGACGAGAGCAACAAGCTGGCCATGATGAAGGTGTTCTGGATCGTCTTGTGGACAGGAGGGAACGGCCACTAGAAGGTGGCCGAACTAATTCCAATAACAACAACAGCAATCCATGTTCAGAACAAGATGTTCTTGCACAGGGTGTTCCAAGCACAGCAGCAGATCCTGGGCCATCAAAGCCCAGAAGAGCACAGAGGCCTAATTCTCTGGATCTTTCAGCCACAAATGTCCTGGATGGCAGCAGTATACAGATAGGTGAGTCAACACAAGATGGCAAATCAGGATCAGGTGAAAAGATCAAGAAACGTGTGAAAACTCCCTATTCTCTTAAGCGGTGGCGCCCCTCCACCTGGGTCATCTCCACTGAATCGCTGGACTGTGAAGTCAACAATAATGGCAGTAACAGGGCAGTTCATTCCAAATCCAGCACTGCTGTTTACCTTGCAGAAGGAGGCACTGCTACAACCATGGTGTCTAAAGATATAG GAATGAACTGTCTG

The nucleic acid sequence encoding the extracellular BMPRII polypeptideis as follows:

(SEQ ID NO: 49) TCGCAGAATCAAGAACGGCTATGTGCGTTTAAAGATCCGTATCAGCAAGACCTTGGGATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCGAAAGGTAGCACCTGCTATGGCCTTTGGGAGAAATCAAAAGGGGACATAAATCTTGTAAAACAAGGATGTTGGTCTCACATTGGAGATCCCCAAGAGTGTCACTATGAAGAATGTGTAGTAACTACCACTCCTCCCTCAATTCAGAATGGAACATACCGTTTCTGCTGTTGTAGCACAGATTTATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGACACAACACCACTCAGTCCACCTCATTCATTTAACCGAGATGAGACA

An alternative isoform of BMPRII, isoform 2 (GenBank: AAA86519.1) is asfollows:

(SEQ ID NO: 71) 1 MTSSLQRPWR VPWLPWTILL VSTAAA SQNQ ERLCAFKDPYQQDLGIGESR 51 ISHENGTILC SKGSTCYGLW EKSKGDINLV KQGCWSHIGD PQECHYEECV 101VTTTPPSIQN GTYRFCCCST DLCNVNFTEN FPPPDTTPLS PPHSFNRDET 151 IIIALASVSVLAVLIVALCF GYRMLTGDRK QGLHSMNMME AAASEPSLDL 201 DNLKLLELIG RGRYGAVYKGSLDERPVAVK VFSFANRQNF INEKNIYRVP 251 LMEHDNIARF IVGDERVTAD GRMEYLLVMEYYPNGSLCKY LSLHTSDWVS 301 SCRLAHSVTR GLAYLHTELP RGDHYKPAIS HRDLNSRNVLVKNDGTCVIS 351 DFGLSMRLTG NRLVRPGEED NAAISEVGTI RYMAPEVLEG AVNLRDCESA401 LKQVDMYALG LIYWEIFMRC TDLFPGESVP EYQMAFQTEV GNHPTFEDMQ 451VLVSREKQRP KFPEAWKENS LAVRSLKETI EDCWDQDAEA RLTAQCAEER 501 MAELMMIWERNKSVSPTVNP MSTAMQNERR

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular BMPRII polypeptide sequence (isoform 2) isas follows:

(SEQ ID NO: 72) SQNQERLCAFKDPYQQDLGIGESRISHENGTILCSKGSTCYGLWEKSKGDINLVKQGCWSHIGDPQECHYEECVVTTTPPSIQNGTYRFCCCSTDLCNVNFTENFPPPDTTPLSPPHSFNRDET

A nucleic acid sequence encoding human BMPRII precursor protein (isoform2) is shown below (SEQ ID NO: 73), corresponding to nucleotides 163-1752of Genbank Reference Sequence U25110.1. The signal sequence isunderlined.

(SEQ ID NO: 73) ATGACTTCCTCGCTGCAGCGGCCCTGGCGGGTGCCCTGGCTACCATGGACCATCCTGCTGGTCAGCACTGCGGCTGCTTCGCAGAATCAAGAACGGCTATGTGCGTTTAAAGATCCGTATCAGCAAGACCTTGGGATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCGAAAGGTAGCACCTGCTATGGCCTTTGGGAGAAATCAAAAGGGGACATAAATCTTGTAAAACAAGGATGTTGGTCTCACATTGGAGATCCCCAAGAGTGTCACTATGAAGAATGTGTAGTAACTACCACTCCTCCCTCAATTCAGAATGGAACATACCGTTTCTGCTGTTGTAGCACAGATTTATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGACACAACACCACTCAGTCCACCTCATTCATTTAACCGAGATGAGACAATAATCATTGCTTTGGCATCAGTCTCTGTATTAGCTGTTTTGATAGTTGCCTTATGCTTTGGATACAGAATGTTGACAGGAGACCGTAAACAAGGTCTTCACAGTATGAACATGATGGAGGCAGCAGCATCCGAACCCTCTCTTGATCTAGATAATCTGAAACTGTTGGAGCTGATTGGCCGAGGTCGATATGGAGCAGTATATAAAGGCTCCTTGGATGAGCGTCCAGTTGCTGTAAAAGTGTTTTCCTTTGCAAACCGTCAGAATTTTATCAACGAAAAGAACATTTACAGAGTGCCTTTGATGGAACATGACAACATTGCCCGCTTTATAGTTGGAGATGAGAGAGTCACTGCAGATGGACGCATGGAATATTTGCTTGTGATGGAGTACTATCCCAATGGATCTTTATGCAAGTATTTAAGTCTCCACACAAGTGACTGGGTAAGCTCTTGCCGTCTTGCTCATTCTGTTACTAGAGGACTGGCTTATCTTCACACAGAATTACCACGAGGAGATCATTATAAACCTGCAATTTCCCATCGAGATTTAAACAGCAGAAATGTCCTAGTGAAAAATGATGGAACCTGTGTTATTAGTGACTTTGGACTGTCCATGAGGCTGACTGGAAATAGACTGGTGCGCCCAGGGGAGGAAGATAATGCAGCCATAAGCGAGGTTGGCACTATCAGATATATGGCACCAGAAGTGCTAGAAGGAGCTGTGAACTTGAGGGACTGTGAATCAGCTTTGAAACAAGTAGACATGTATGCTCTTGGACTAATCTATTGGGAGATATTTATGAGATGTACAGACCTCTTCCCAGGGGAATCCGTACCAGAGTACCAGATGGCTTTTCAGACAGAGGTTGGAAACCATCCCACTTTTGAGGATATGCAGGTTCTCGTGTCTAGGGAAAAACAGAGACCCAAGTTCCCAGAAGCCTGGAAAGAAAATAGCCTGGCAGTGAGGTCACTCAAGGAGACAATCGAAGACTGTTGGGACCAGGATGCAGAGGCTCGGCTTACTGCACAGTGTGCTGAGGAAAGGATGGCTGAACTTATGATGATTTGGGAAAGAAACAAATCTGTGAGCCCAACAGTCAATCCAATGTCTACTGCTATGCAGAATGAACGTAGG

A nucleic acid sequence encoding an extracellular BMPRII polypeptide(isoform 2) is as follows:

(SEQ ID NO: 74) TCGCAGAATCAAGAACGGCTATGTGCGTTTAAAGATCCGTATCAGCAAGACCTTGGGATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCGAAAGGTAGCACCTGCTATGGCCTTTGGGAGAAATCAAAAGGGGACATAAATCTTGTAAAACAAGGATGTTGGTCTCACATTGGAGATCCCCAAGAGTGTCACTATGAAGAATGTGTAGTAACTACCACTCCTCCCTCAATTCAGAATGGAACATACCGTTTCTGCTGTTGTAGCACAGATTTATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGACACAACACCACTCAGTCCACCTCATTCATTTAACCGAGATGAGACA

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one BMPRII polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, BMPRII polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising a BMPRII polypeptide and uses thereof) are soluble (e.g., anextracellular domain of BMPRII). In other preferred embodiments, BMPRIIpolypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one BMPRII polypeptide that is at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence of SEQ ID NO: 46, 47, 71, 72, 107, 109, 405, or 406. Insome embodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one BMPRII polypeptide thatis at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO: 46, 47, 71, 72, 107, 109, 405, or406.

In certain aspects, the present disclosure relates to protein complexesthat comprise an MISRII polypeptide. As used herein, the term “MISRII”refers to a family of Müllerian inhibiting substance receptor type II(MISRII) proteins from any species and variants derived from such MISRIIproteins by mutagenesis or other modification. Reference to MISRIIherein is understood to be a reference to any one of the currentlyidentified forms. Members of the MISRII family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain with a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The term “MISRII polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an MISRII family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all MISRII-related polypeptides described herein is based onthe numbering of the human MISRII precursor protein sequence below (SEQID NO: 50), unless specifically designated otherwise.

The canonical human MISRII precursor protein sequence (NCBI Ref SeqNP_065434.1) is as follows:

(SEQ ID NO: 50) 1 MLGSLGLWAL LPTAVEA PPN RRTCVFFEAP GVRGSTKTLGELLDTGTELP 51 RAIRCLYSRC CFGIWNLTQD RAQVEMQGCR DSDEPGCESL HCDPSPRAHP 101SPGSTLFTCS CGTDFCNANY SHLPPPGSPG TPGSQGPQAA PGESIWMALV 151 LLGLFLLLLLLLGSIILALL QRKNYRVRGE PVPEPRPDSG RDWSVELQEL 201 PELCFSQVIR EGGHAVVWAGQLQGKLVAIK AFPPRSVAQF QAERALYELP 251 GLQHDHIVRF ITASRGGPGR LLSGPLLVLELHPKGSLCHY LTQYTSDWGS 301 SLRMALSLAQ GLAFLHEERW QNGQYKPGIA HRDLSSQNVLIREDGSCAIG 351 DLGLALVLPG LTQPPAWTPT QPQGPAAIME AGTQRYMAPE LLDKTLDLQD401 WGMALRRADI YSLALLLWEI LSRCPDLRPD SSPPPFQLAY EAELGNTPTS 451DELWALAVQE RRRPYIPSTW RCFATDPDGL RELLEDCWDA DPEARLTAEC 501 VQQRLAALAHPQESHPFPES CPRGCPPLCP EDCTSIPAPT ILPCRPQRSA 551 CHFSVQQGPC SRNPQPACTLSPV

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular MISRII polypeptide sequence is as follows:

(SEQ ID NO: 51) PPNRRTCVFFEAPGVRGSTKTLGELLDTGTELPRAIRCLYSRCCFGIWNLTQDRAQVEMQGCRDSDEPGCESLHCDPSPRAHPSPGSTLFTCSCGTDFCNANYSHLPPPGSPGTPGSQGPQAAPGESIWMAL

A nucleic acid sequence encoding the MISRII precursor protein is shownbelow (SEQ ID NO: 52), corresponding to nucleotides 81-1799 of GenbankReference Sequence NM_020547.2. The signal sequence is underlined.

(SEQ ID NO: 52) ATGCTAGGGTCTTTGGGGCTTTGGGCATTACTTCCCACAGCTGTGGAAGC ACCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTGGTGCTGCTGGGGCTGTTCCTCCTCCTCCTGCTGCTGCTGGGCAGCATCATCTTGGCCCTGCTACAGCGAAAGAACTACAGAGTGCGAGGTGAGCCAGTGCCAGAGCCAAGGCCAGACTCAGGCAGGGACTGGAGTGTGGAGCTGCAGGAGCTGCCTGAGCTGTGTTTCTCCCAGGTAATCCGGGAAGGAGGTCATGCAGTGGTTTGGGCCGGGCAGCTGCAAGGAAAACTGGTTGCCATCAAGGCCTTCCCACCGAGGTCTGTGGCTCAGTTCCAAGCTGAGAGAGCATTGTACGAACTTCCAGGCCTACAGCACGACCACATTGTCCGATTTATCACTGCCAGCCGGGGGGGTCCTGGCCGCCTGCTCTCTGGGCCCCTGCTGGTACTGGAACTGCATCCCAAGGGCTCCCTGTGCCACTACTTGACCCAGTACACCAGTGACTGGGGAAGTTCCCTGCGGATGGCACTGTCCCTGGCCCAGGGCCTGGCATTTCTCCATGAGGAGCGCTGGCAGAATGGCCAATATAAACCAGGTATTGCCCACCGAGATCTGAGCAGCCAGAATGTGCTCATTCGGGAAGATGGATCGTGTGCCATTGGAGACCTGGGCCTTGCCTTGGTGCTCCCTGGCCTCACTCAGCCCCCTGCCTGGACCCCTACTCAACCACAAGGCCCAGCTGCCATCATGGAAGCTGGCACCCAGAGGTACATGGCACCAGAGCTCTTGGACAAGACTCTGGACCTACAGGATTGGGGCATGGCCCTCCGACGAGCTGATATTTACTCTTTGGCTCTGCTCCTGTGGGAGATACTGAGCCGCTGCCCAGATTTGAGGCCTGACAGCAGTCCACCACCCTTCCAACTGGCCTATGAGGCAGAACTGGGCAATACCCCTACCTCTGATGAGCTATGGGCCTTGGCAGTGCAGGAGAGGAGGCGTCCCTACATCCCATCCACCTGGCGCTGCTTTGCCACAGACCCTGATGGGCTGAGGGAGCTCCTAGAAGACTGTTGGGATGCAGACCCAGAAGCACGGCTGACAGCTGAGTGTGTACAGCAGCGCCTGGCTGCCTTGGCCCATCCTCAAGAGAGCCACCCCTTTCCAGAGAGCTGTCCACGTGGCTGCCCACCTCTCTGCCCAGAAGACTGTACTTCAATTCCTGCCCCTACCATCCTCCCCTGTAGGCCTCAGCGGAGTGCCTGCCACTTCAGCGTTCAGCAAGGCCCTTGTTCCAGGAATCCTCAGCCTGC CTGTACCCTTTCTCCTGTG

A nucleic acid sequence encoding the extracellular human MISRIIpolypeptide is as follows:

(SEQ ID NO: 53) CCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTG

An alternative isoform of the human MISRII precursor protein sequence,isoform 2 (NCBI Ref Seq NP_001158162.1), is as follows:

(SEQ ID NO: 75) 1 MLGSLGLWAL LPTAVEA PPN RRTCVFFEAP GVRGSTKTLGELLDTGTELP 051 RAIRCLYSRC CFGIWNLTQD RAQVEMQGCR DSDEPGCESL HCDPSPRAHP101 SPGSTLFTCS CGTDFCNANY SHLPPPGSPG TPGSQGPQAA PGESIWMALV 151LLGLFLLLLL LLGSIILALL QRKNYRVRGE PVPEPRPDSG RDWSVELQEL 201 PELCFSQVIREGGHAVVWAG QLQGKLVAIK AFPPRSVAQF QAERALYELP 251 GLQHDHIVRF ITASRGGPGRLLSGPLLVLE LHPKGSLCHY LTQYTSDWGS 301 SLRMALSLAQ GLAFLHEERW QNGQYKPGIAHRDLSSQNVL IREDGSCAIG 351 DLGLALVLPG LTQPPAWTPT QPQGPAAIME AGTQRYMAPELLDKTLDLQD 401 WGMALRRADI YSLALLLWEI LSRCPDLRPA VHHPSNWPMR QNWAIPLPLM451 SYGPWQCRRG GVPTSHPPGA ALPQTLMG

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular MISRII polypeptide sequence (isoform 2) isas follows:

(SEQ ID NO: 76) PPNRRTCVFFEAPGVRGSTKTLGELLDTGTELPRAIRCLYSRCCFGIWNLTQDRAQVEMQGCRDSDEPGCESLHCDPSPRAHPSPGSTLFTCSCGTDFCNANYSHLPPPGSPGTPGSQGPQAAPGESIWMAL

A nucleic acid sequence encoding the MISRII precursor protein (isoform2) is shown below (SEQ ID NO: 77), corresponding to nucleotides 81-1514of Genbank Reference Sequence NM_001164690.1. The signal sequence isunderlined.

(SEQ ID NO: 77) ATGCTAGGGTCTTTGGGGCTTTGGGCATTACTTCCCACAGCTGTGGAAGCACCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTGGTGCTGCTGGGGCTGTTCCTCCTCCTCCTGCTGCTGCTGGGCAGCATCATCTTGGCCCTGCTACAGCGAAAGAACTACAGAGTGCGAGGTGAGCCAGTGCCAGAGCCAAGGCCAGACTCAGGCAGGGACTGGAGTGTGGAGCTGCAGGAGCTGCCTGAGCTGTGTTTCTCCCAGGTAATCCGGGAAGGAGGTCATGCAGTGGTTTGGGCCGGGCAGCTGCAAGGAAAACTGGTTGCCATCAAGGCCTTCCCACCGAGGTCTGTGGCTCAGTTCCAAGCTGAGAGAGCATTGTACGAACTTCCAGGCCTACAGCACGACCACATTGTCCGATTTATCACTGCCAGCCGGGGGGGTCCTGGCCGCCTGCTCTCTGGGCCCCTGCTGGTACTGGAACTGCATCCCAAGGGCTCCCTGTGCCACTACTTGACCCAGTACACCAGTGACTGGGGAAGTTCCCTGCGGATGGCACTGTCCCTGGCCCAGGGCCTGGCATTTCTCCATGAGGAGCGCTGGCAGAATGGCCAATATAAACCAGGTATTGCCCACCGAGATCTGAGCAGCCAGAATGTGCTCATTCGGGAAGATGGATCGTGTGCCATTGGAGACCTGGGCCTTGCCTTGGTGCTCCCTGGCCTCACTCAGCCCCCTGCCTGGACCCCTACTCAACCACAAGGCCCAGCTGCCATCATGGAAGCTGGCACCCAGAGGTACATGGCACCAGAGCTCTTGGACAAGACTCTGGACCTACAGGATTGGGGCATGGCCCTCCGACGAGCTGATATTTACTCTTTGGCTCTGCTCCTGTGGGAGATACTGAGCCGCTGCCCAGATTTGAGGCCTGCAGTCCACCACCCTTCCAACTGGCCTATGAGGCAGAACTGGGCAATACCCCTACCTCTGATGAGCTATGGGCCTTGGCAGTGCAGGAGAGGAGGCGTCCCTACATCCCATCCACCTGGCGCTGCTTTGCCACAGACCCTGATGGGC

The nucleic acid sequence encoding processed soluble (extracellular)human MISRII polypeptide (isoform 2) is as follows:

(SEQ ID NO: 78) CCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTG

An alternative isoform of the human MISRII precursor protein sequence,isoform 3 (NCBI Ref Seq NP_001158163.1), is as follows:

(SEQ ID NO: 79) 1 MLGSLGLWAL LPTAVEA PPN RRTCVFFEAP GVRGSTKTLGELLDTGTELP 51 RAIRCLYSRC CFGIWNLTQD RAQVEMQGCR DSDEPGCESL HCDPSPRAHP 101SPGSTLFTCS CGTDFCNANY SHLPPPGSPG TPGSQGPQAA PGESIWMALV 151 LLGLFLLLLLLLGSIILALL QRKNYRVRGE PVPEPRPDSG RDWSVELQEL 201 PELCFSQVIR EGGHAVVWAGQLQGKLVAIK AFPPRSVAQF QAERALYELP 251 GLQHDHIVRF ITASRGGPGR LLSGPLLVLELHPKGSLCHY LTQYTSDWGS 301 SLRMALSLAQ GLAFLHEERW QNGQYKPGIA HRDLSSQNVLIREDGSCAIG 351 DLGLALVLPG LTQPPAWTPT QPQGPAAIME DPDGLRELLE DCWDADPEAR401 LTAECVQQRL AALAHPQESH PFPESCPRGC PPLCPEDCTS IPAPTILPCR 451PQRSACHFSV QQGPCSRNPQ PACTLSPV

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular MISRII polypeptide sequence (isoform 3) isas follows:

(SEQ ID NO: 80) PPNRRTCVFFEAPGVRGSTKTLGELLDTGTELPRAIRCLYSRCCFGIWNLTQDRAQVEMQGCRDSDEPGCESLHCDPSPRAHPSPGSTLFTCSCGTDFCNANYSHLPPPGSPGTPGSQGPQAAPGESIWMAL

A nucleic acid sequence encoding human MISRII precursor protein (isoform3) is shown below (SEQ ID NO: 81), corresponding to nucleotides 81-1514of Genbank Reference Sequence NM_001164691.1. The signal sequence isunderlined.

(SEQ ID NO: 81) ATGCTAGGGTCTTTGGGGCTTTGGGCATTACTTCCCACAGCTGTGGAAGCACCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTGGTGCTGCTGGGGCTGTTCCTCCTCCTCCTGCTGCTGCTGGGCAGCATCATCTTGGCCCTGCTACAGCGAAAGAACTACAGAGTGCGAGGTGAGCCAGTGCCAGAGCCAAGGCCAGACTCAGGCAGGGACTGGAGTGTGGAGCTGCAGGAGCTGCCTGAGCTGTGTTTCTCCCAGGTAATCCGGGAAGGAGGTCATGCAGTGGTTTGGGCCGGGCAGCTGCAAGGAAAACTGGTTGCCATCAAGGCCTTCCCACCGAGGTCTGTGGCTCAGTTCCAAGCTGAGAGAGCATTGTACGAACTTCCAGGCCTACAGCACGACCACATTGTCCGATTTATCACTGCCAGCCGGGGGGGTCCTGGCCGCCTGCTCTCTGGGCCCCTGCTGGTACTGGAACTGCATCCCAAGGGCTCCCTGTGCCACTACTTGACCCAGTACACCAGTGACTGGGGAAGTTCCCTGCGGATGGCACTGTCCCTGGCCCAGGGCCTGGCATTTCTCCATGAGGAGCGCTGGCAGAATGGCCAATATAAACCAGGTATTGCCCACCGAGATCTGAGCAGCCAGAATGTGCTCATTCGGGAAGATGGATCGTGTGCCATTGGAGACCTGGGCCTTGCCTTGGTGCTCCCTGGCCTCACTCAGCCCCCTGCCTGGACCCCTACTCAACCACAAGGCCCAGCTGCCATCATGGAAGACCCTGATGGGCTGAGGGAGCTCCTAGAAGACTGTTGGGATGCAGACCCAGAAGCACGGCTGACAGCTGAGTGTGTACAGCAGCGCCTGGCTGCCTTGGCCCATCCTCAAGAGAGCCACCCCTTTCCAGAGAGCTGTCCACGTGGCTGCCCACCTCTCTGCCCAGAAGACTGTACTTCAATTCCTGCCCCTACCATCCTCCCCTGTAGGCCTCAGCGGAGTGCCTGCCACTTCAGCGTTCAGCAAGGCCCTTGTTCCAGGAATCCTCAGCCTGCCTGTACCCTTTCTCCTGTG

A nucleic acid sequence encoding processed soluble (extracellular) humanMISRII polypeptide (isoform 3) is as follows:

(SEQ ID NO: 82) CCCCCAAACAGGCGAACCTGTGTGTTCTTTGAGGCCCCTGGAGTGCGGGGAAGCACAAAGACACTGGGAGAGCTGCTAGATACAGGCACAGAGCTCCCCAGAGCTATCCGCTGCCTCTACAGCCGCTGCTGCTTTGGGATCTGGAACCTGACCCAAGACCGGGCACAGGTGGAAATGCAAGGATGCCGAGACAGTGATGAGCCAGGCTGTGAGTCCCTCCACTGTGACCCAAGTCCCCGAGCCCACCCCAGCCCTGGCTCCACTCTCTTCACCTGCTCCTGTGGCACTGACTTCTGCAATGCCAATTACAGCCATCTGCCTCCTCCAGGGAGCCCTGGGACTCCTGGCTCCCAGGGTCCCCAGGCTGCCCCAGGTGAGTCCATCTGGATGGCACTG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one MISRII polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, MISRII polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising a MISRII polypeptide and uses thereof) are soluble (e.g., anextracellular domain of MISRII). In other preferred embodiments, MISRIIpolypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one MISRII polypeptide that is at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence of SEQ ID NOs: 50, 51, 75, 76, 79, 80, 110, 112, 407, or408. In some embodiments, single-arm heteromultimer complexes of thedisclosure consist or consist essentially of at least one MISRIIpolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NOs: 50, 51, 75, 76,79, 80, 110, 112, 407, or 408.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK1 polypeptide. As used herein, the term “ALK1”refers to a family of activin receptor-like kinase-1 proteins from anyspecies and variants derived from such ALK1 proteins by mutagenesis orother modification. Reference to ALK1 herein is understood to be areference to any one of the currently identified forms. Members of theALK1 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK1 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK1 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK1-related polypeptides described herein is based on thenumbering of the human ALK1 precursor protein sequence below (SEQ ID NO:14), unless specifically designated otherwise.

The human ALK1 precursor protein sequence (NCBI Ref Seq NP_000011.2) isas follows:

(SEQ ID NO: 14) 1 MTLGSPRKGL LMLLMALVTQ G DPVKPSRGP LVTCTCESPHCKGPTCRGAW 51 CTVVLVREEG RHPQEHRGCG NLHRELCRGR PTEFVNHYCC DSHLCNHNVS 101LVLEATQPPS EQPGTDGQLA LILGPVLALL ALVALGVLGL WHVRRRQEKQ 151 RGLHSELGESSLILKASEQG DSMLGDLLDS DCTTGSGSGL PFLVQRTVAR 201 QVALVECVGK GRYGEVWRGLWHGESVAVKI FSSRDEQSWF RETEIYNTVL 251 LRHDNILGFI ASDMTSRNSS TQLWLITHYHEHGSLYDFLQ RQTLEPHLAL 301 RLAVSAACGL AHLHVEIFGT QGKPAIAHRD FKSRNVLVKSNLQCCIADLG 351 LAVMHSQGSD YLDIGNNPRV GTKRYMAPEV LDEQIRTDCF ESYKWTDIWA401 FGLVLWEIAR RTIVNGIVED YRPPFYDVVP NDPSFEDMKK VVCVDQQTPT 451IPNRLAADPV LSGLAQMMRE CWYPNPSARL TALRIKKTLQ KISNSPEKPK 501 VIQ

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracelluar ALK1 polypeptide sequence is as follows:

(SEQ ID NO: 15) DPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQ

A nucleic acid sequence encoding human ALK1 precursor protein is shownbelow (SEQ ID NO: 16), corresponding to nucleotides 284-1792 of GenbankReference Sequence NM_000020.2. The signal sequence is underlined.

(SEQ ID NO: 16) ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGA GACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGCTGGCCCTGATCCTGGGCCCCGTGCTGGCCTTGCTGGCCCTGGTGGCCCTGGGTGTCCTGGGCCTGTGGCATGTCCGACGGAGGCAGGAGAAGCAGCGTGGCCTGCACAGCGAGCTGGGAGAGTCCAGTCTCATCCTGAAAGCATCTGAGCAGGGCGACAGCATGTTGGGGGACCTCCTGGACAGTGACTGCACCACAGGGAGTGGCTCAGGGCTCCCCTTCCTGGTGCAGAGGACAGTGGCACGGCAGGTTGCCTTGGTGGAGTGTGTGGGAAAAGGCCGCTATGGCGAAGTGTGGCGGGGCTTGTGGCACGGTGAGAGTGTGGCCGTCAAGATCTTCTCCTCGAGGGATGAACAGTCCTGGTTCCGGGAGACTGAGATCTATAACACAGTGTTGCTCAGACACGACAACATCCTAGGCTTCATCGCCTCAGACATGACCTCCCGCAACTCGAGCACGCAGCTGTGGCTCATCACGCACTACCACGAGCACGGCTCCCTCTACGACTTTCTGCAGAGACAGACGCTGGAGCCCCATCTGGCTCTGAGGCTAGCTGTGTCCGCGGCATGCGGCCTGGCGCACCTGCACGTGGAGATCTTCGGTACACAGGGCAAACCAGCCATTGCCCACCGCGACTTCAAGAGCCGCAATGTGCTGGTCAAGAGCAACCTGCAGTGTTGCATCGCCGACCTGGGCCTGGCTGTGATGCACTCACAGGGCAGCGATTACCTGGACATCGGCAACAACCCGAGAGTGGGCACCAAGCGGTACATGGCACCCGAGGTGCTGGACGAGCAGATCCGCACGGACTGCTTTGAGTCCTACAAGTGGACTGACATCTGGGCCTTTGGCCTGGTGCTGTGGGAGATTGCCCGCCGGACCATCGTGAATGGCATCGTGGAGGACTATAGACCACCCTTCTATGATGTGGTGCCCAATGACCCCAGCTTTGAGGACATGAAGAAGGTGGTGTGTGTGGATCAGCAGACCCCCACCATCCCTAACCGGCTGGCTGCAGACCCGGTCCTCTCAGGCCTAGCTCAGATGATGCGGGAGTGCTGGTACCCAAACCCCTCTGCCCGACTCACCGCGCTGCGGATCAAGAAGACACTACAAAAAATTAGCAACAGTCCAGAGAAGCCTAAA GTGATTCAA

A nucleic acid sequence encoding processed extracelluar ALK1 polypeptideis as follows:

(SEQ ID NO: 17) GACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK1 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK1 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK1 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK1). In other preferred embodiments, ALK1polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK1 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 14, 15, 116, 118, 411, or 412. In someembodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK1 polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 14, 15, 116, 118, 411, or 412.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK2 polypeptide. As used herein, the term “ALK2”refers to a family of activin receptor-like kinase-2 proteins from anyspecies and variants derived from such ALK2 proteins by mutagenesis orother modification. Reference to ALK2 herein is understood to be areference to any one of the currently identified forms. Members of theALK2 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK2 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK2 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK2-related polypeptides described herein is based on thenumbering of the human ALK2 precursor protein sequence below (SEQ ID NO:18), unless specifically designated otherwise.

The human ALK2 precursor protein sequence (NCBI Ref Seq NP_001096.1) isas follows:

(SEQ ID NO: 18) 1 MVDGVMILPV LIMIALPSPS MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG51 QQCFSSLSIN DGFHVYQKGC FQVYEQGKMT CKTPPSPGQA VECCQGDWCN 101 RNITAQLPTKGKSFPGTQNF HLEVGLIILS VVFAVCLLAC LLGVALRKFK 151 RRNQERLNPR DVEYGTIEGLITTNVGDSTL ADLLDHSCTS GSGSGLPFLV 201 QRTVARQITL LECVGKGRYG EVWRGSWQGENVAVKIFSSR DEKSWFRETE 251 LYNTVMLRHE NILGFIASDM TSRHSSTQLW LITHYHEMGSLYDYLQLTTL 301 DTVSCLRIVL SIASGLAHLH IEIFGTQGKP AIAHRDLKSK NILVKKNGQC351 CIADLGLAVM HSQSTNQLDV GNNPRVGTKR YMAPEVLDET IQVDCFDSYK 401RVDIWAFGLV LWEVARRMVS NGIVEDYKPP FYDVVPNDPS FEDMRKVVCV 451 DQQRPNIPNRWFSDPTLTSL AKLMKECWYQ NPSARLTALR IKKTLTKIDN 501 SLDKLKTDC

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK2 polypeptide sequence is as follows:

(SEQ ID NO: 19) MEDEKPKVNPKLYMCVCEGLSCGNEDHCEGQQCFSSLSINDGFHVYQKGCFQVYEQGKMTCKTPPSPGQAVECCQGDWCNRNITAQLPTKGKSFPGT QNFHLE

A nucleic acid sequence encoding human ALK2 precursor protein is shownbelow (SEQ ID NO: 20), corresponding to nucleotides 431-1957 of GenbankReference Sequence NM_001105.4. The signal sequence is underlined.

(SEQ ID NO: 20) ATGGTAGATGGAGTGATGATTCTTCCTGTGCTTATCATGATTGCTCTCCCCTCCCCTAGT ATGGAAGATGAGAAGCCCAAGGTCAACCCCAAACTCTACATGTGTGTGTGTGAAGGTCTCTCCTGCGGTAATGAGGACCACTGTGAAGGCCAGCAGTGCTTTTCCTCACTGAGCATCAACGATGGCTTCCACGTCTACCAGAAAGGCTGCTTCCAGGTTTATGAGCAGGGAAAGATGACCTGTAAGACCCCGCCGTCCCCTGGCCAAGCCGTGGAGTGCTGCCAAGGGGACTGGTGTAACAGGAACATCACGGCCCAGCTGCCCACTAAAGGAAAATCCTTCCCTGGAACACAGAATTTCCACTTGGAGGTTGGCCTCATTATTCTCTCTGTAGTGTTCGCAGTATGTCTTTTAGCCTGCCTGCTGGGAGTTGCTCTCCGAAAATTTAAAAGGCGCAACCAAGAACGCCTCAATCCCCGAGACGTGGAGTATGGCACTATCGAAGGGCTCATCACCACCAATGTTGGAGACAGCACTTTAGCAGATTTATTGGATCATTCGTGTACATCAGGAAGTGGCTCTGGTCTTCCTTTTCTGGTACAAAGAACAGTGGCTCGCCAGATTACACTGTTGGAGTGTGTCGGGAAAGGCAGGTATGGTGAGGTGTGGAGGGGCAGCTGGCAAGGGGAGAATGTTGCCGTGAAGATCTTCTCCTCCCGTGATGAGAAGTCATGGTTCAGGGAAACGGAATTGTACAACACTGTGATGCTGAGGCATGAAAATATCTTAGGTTTCATTGCTTCAGACATGACATCAAGACACTCCAGTACCCAGCTGTGGTTAATTACACATTATCATGAAATGGGATCGTTGTACGACTATCTTCAGCTTACTACTCTGGATACAGTTAGCTGCCTTCGAATAGTGCTGTCCATAGCTAGTGGTCTTGCACATTTGCACATAGAGATATTTGGGACCCAAGGGAAACCAGCCATTGCCCATCGAGATTTAAAGAGCAAAAATATTCTGGTTAAGAAGAATGGACAGTGTTGCATAGCAGATTTGGGCCTGGCAGTCATGCATTCCCAGAGCACCAATCAGCTTGATGTGGGGAACAATCCCCGTGTGGGCACCAAGCGCTACATGGCCCCCGAAGTTCTAGATGAAACCATCCAGGTGGATTGTTTCGATTCTTATAAAAGGGTCGATATTTGGGCCTTTGGACTTGTTTTGTGGGAAGTGGCCAGGCGGATGGTGAGCAATGGTATAGTGGAGGATTACAAGCCACCGTTCTACGATGTGGTTCCCAATGACCCAAGTTTTGAAGATATGAGGAAGGTAGTCTGTGTGGATCAACAAAGGCCAAACATACCCAACAGATGGTTCTCAGACCCGACATTAACCTCTCTGGCCAAGCTAATGAAAGAATGCTGGTATCAAAATCCATCCGCAAGACTCACAGCACTGCGTATCAAAAAGACTTTGACCAAAATTGATAATTCCCTCGACAAATTGAAAACTGACTGT

A nucleic acid sequence encoding the extracellular ALK2 polypeptide isas follows:

(SEQ ID NO: 21) ATGGAAGATGAGAAGCCCAAGGTCAACCCCAAACTCTACATGTGTGTGTGTGAAGGTCTCTCCTGCGGTAATGAGGACCACTGTGAAGGCCAGCAGTGCTTTTCCTCACTGAGCATCAACGATGGCTTCCACGTCTACCAGAAAGGCTGCTTCCAGGTTTATGAGCAGGGAAAGATGACCTGTAAGACCCCGCCGTCCCCTGGCCAAGCCGTGGAGTGCTGCCAAGGGGACTGGTGTAACAGGAACATCACGGCCCAGCTGCCCACTAAAGGAAAATCCTTCCCTGGAACACAGAATTTC CACTTGGAG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK2 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK2 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK2 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK2). In other preferred embodiments, ALK2polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK2 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 18, 19, 119, 121, 413, or 414. In someembodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK2 polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 18, 19, 119, 121, 413, or 414.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK3 polypeptide. As used herein, the term “ALK3”refers to a family of activin receptor-like kinase-3 proteins from anyspecies and variants derived from such ALK3 proteins by mutagenesis orother modification. Reference to ALK3 herein is understood to be areference to any one of the currently identified forms. Members of theALK3 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK3 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK3 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK3-related polypeptides described herein is based on thenumbering of the human ALK3 precursor protein sequence below (SEQ ID NO:22), unless specifically designated otherwise.

The human ALK3 precursor protein sequence (NCBI Ref Seq NP_004320.2) isas follows:

(SEQ ID NO: 22) 1 MPQLYIYIRL LGAYLFIISR VQG QNLDSML HGTGMKSDSDQKKSENGVTL APEDTLPFLK 61 CYCSGHCPDD AINNTCITNG HCFAIIEEDD QGETTLASGCMKYEGSDFQC KDSPKAQLRR 121 TIECCRTNLC NQYLQPTLPP VVIGPFFDGS IRWLVLLISMAVCIIAMIIF SSCFCYKHYC 181 KSISSRRRYN RDLEQDEAFI PVGESLKDLI DQSQSSGSGSGLPLLVQRTI AKQIQMVRQV 241 GKGRYGEVWM GKWRGEKVAV KVFFTTEEAS WFRETEIYQTVLMRHENILG FIAADIKGTG 301 SWTQLYLITD YHENGSLYDF LKCATLDTRA LLKLAYSAACGLCHLHTEIY GTQGKPAIAH 361 RDLKSKNILI KKNGSCCIAD LGLAVKFNSD TNEVDVPLNTRVGTKRYMAP EVLDESLNKN 421 HFQPYIMADI YSFGLIIWEM ARRCITGGIV EEYQLPYYNMVPSDPSYEDM REVVCVKRLR 481 PIVSNRWNSD ECLRAVLKLM SECWAHNPAS RLTALRIKKTLAKMVESQDV KI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK3 polypeptide sequence is as follows:

(SEQ ID NO: 23) 1 QNLDSMLHGT GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAINNTCITNGHCF 61 AIIEEDDQGE TTLASGCMKY EGSDFQCKDS PKAQLRRTIE CCRTNLCNQYLQPTLPPVVI 121 GPFFDGSIR

A nucleic acid sequence encoding human ALK3 precursor protein is shownbelow (SEQ ID NO: 24), corresponding to nucleotides 549-2144 of GenbankReference Sequence NM_004329.2. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 24) 1 ATGCCTCAGC TATACATTTA CATCAGATTA TTGGGAGCCT ATTTGTTCATCATTTCTCGT 61 GTTCAAGGA C AGAATCTGGA TAGTATGCTT CATGGCACTG GGATGAAATCAGACTCCGAC 121 CAGAAAAAGT CAGAAAATGG AGTAACCTTA GCACCAGAGG ATACCTTGCCTTTTTTAAAG 181 TGCTATTGCT CAGGGCACTG TCCAGATGAT GCTATTAATA ACACATGCATAACTAATGGA 241 CATTGCTTTG CCATCATAGA AGAAGATGAC CAGGGAGAAA CCACATTAGCTTCAGGGTGT 301 ATGAAATATG AAGGATCTGA TTTTCAGTGC AAAGATTCTC CAAAAGCCCAGCTACGCCGG 361 ACAATAGAAT GTTGTCGGAC CAATTTATGT AACCAGTATT TGCAACCCACACTGCCCCCT 421 GTTGTCATAG GTCCGTTTTT TGATGGCAGC ATTCGATGGC TGGTTTTGCTCATTTCTATG 481 GCTGTCTGCA TAATTGCTAT GATCATCTTC TCCAGCTGCT TTTGTTACAAACATTATTGC 541 AAGAGCATCT CAAGCAGACG TCGTTACAAT CGTGATTTGG AACAGGATGAAGCATTTATT 601 CCAGTTGGAG AATCACTAAA AGACCTTATT GACCAGTCAC AAAGTTCTGGTAGTGGGTCT 661 GGACTACCTT TATTGGTTCA GCGAACTATT GCCAAACAGA TTCAGATGGTCCGGCAAGTT 721 GGTAAAGGCC GATATGGAGA AGTATGGATG GGCAAATGGC GTGGCGAAAAAGTGGCGGTG 781 AAAGTATTCT TTACCACTGA AGAAGCCAGC TGGTTTCGAG AAACAGAAATCTACCAAACT 841 GTGCTAATGC GCCATGAAAA CATACTTGGT TTCATAGCGG CAGACATTAAAGGTACAGGT 901 TCCTGGACTC AGCTCTATTT GATTACTGAT TACCATGAAA ATGGATCTCTCTATGACTTC 961 CTGAAATGTG CTACACTGGA CACCAGAGCC CTGCTTAAAT TGGCTTATTCAGCTGCCTGT 1021 GGTCTGTGCC ACCTGCACAC AGAAATTTAT GGCACCCAAG GAAAGCCCGCAATTGCTCAT 1081 CGAGACCTAA AGAGCAAAAA CATCCTCATC AAGAAAAATG GGAGTTGCTGCATTGCTGAC 1141 CTGGGCCTTG CTGTTAAATT CAACAGTGAC ACAAATGAAG TTGATGTGCCCTTGAATACC 1201 AGGGTGGGCA CCAAACGCTA CATGGCTCCC GAAGTGCTGG ACGAAAGCCTGAACAAAAAC 1261 CACTTCCAGC CCTACATCAT GGCTGACATC TACAGCTTCG GCCTAATCATTTGGGAGATG 1321 GCTCGTCGTT GTATCACAGG AGGGATCGTG GAAGAATACC AATTGCCATATTACAACATG 1381 GTACCGAGTG ATCCGTCATA CGAAGATATG CGTGAGGTTG TGTGTGTCAAACGTTTGCGG 1441 CCAATTGTGT CTAATCGGTG GAACAGTGAT GAATGTCTAC GAGCAGTTTTGAAGCTAATG 1501 TCAGAATGCT GGGCCCACAA TCCAGCCTCC AGACTCACAG CATTGAGAATTAAGAAGACG 1561 CTTGCCAAGA TGGTTGAATC CCAAGATGTA AAAATC

A nucleic acid sequence encoding the extracelluar human ALK3 polypeptideis as follows:

(SEQ ID NO: 25) 1 CAGAATCTGG ATAGTATGCT TCATGGCACT GGGATGAAAT CAGACTCCGACCAGAAAAAG 61 TCAGAAAATG GAGTAACCTT AGCACCAGAG GATACCTTGC CTTTTTTAAAGTGCTATTGC 121 TCAGGGCACT GTCCAGATGA TGCTATTAAT AACACATGCA TAACTAATGGACATTGCTTT 181 GCCATCATAG AAGAAGATGA CCAGGGAGAA ACCACATTAG CTTCAGGGTGTATGAAATAT 241 GAAGGATCTG ATTTTCAGTG CAAAGATTCT CCAAAAGCCC AGCTACGCCGGACAATAGAA 301 TGTTGTCGGA CCAATTTATG TAACCAGTAT TTGCAACCCA CACTGCCCCCTGTTGTCATA 361 GGTCCGTTTT TTGATGGCAG CATTCGA

A general formula for an active (e.g., ligand binding) ALK3 polypeptideis one that comprises a polypeptide that begins at any amino acidposition 25-31 (i.e., position 25, 26, 27, 28, 29, 30, or 31) of SEQ IDNO: 22 and ends at any amino acid position 140-152 of SEQ ID NO: 22(i.e., 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, or152). See U.S. Pat. No. 8,338,377, the teachings of which areincorporated herein by reference in their entirety.

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK3 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK3 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK3 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK3). In other preferred embodiments, ALK3polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK3 polypeptide that comprises,consists, or consists essentially of an amino acid beginning at anyamino acid position 25-31 (i.e., position 25, 26, 27, 28, 29, 30, or 31)of SEQ ID NO: 22 and ending at any amino acid position 140-153 of SEQ IDNO: 22 (i.e., 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, or 152) of SEQ ID NO: 22. In some embodiments, single-armheteromultimer complexes of the disclosure comprise at least one ALK3polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NO: 22, 23, 122, 124,415, or 416. In some embodiments, single-arm heteromultimer complexes ofthe disclosure consist or consist essentially of at least one ALK3polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NO: 22, 23, 122, 124,415, or 416.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK4 polypeptide. As used herein, the term “ALK4”refers to a family of activin receptor-like kinase-4 proteins from anyspecies and variants derived from such ALK4 proteins by mutagenesis orother modification. Reference to ALK4 herein is understood to be areference to any one of the currently identified forms. Members of theALK4 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK4 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK4 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK4-related polypeptides described herein is based on thenumbering of the human ALK4 precursor protein sequence below (SEQ ID NO:26), unless specifically designated otherwise.

The protein sequence of canonical human ALK4 precursor (isoform A, NCBIRef Seq NP_004293) is as follows:

(SEQ ID NO: 26) 1 MAESAGASSF FPLVVLLLAG SGG SGPRGVQ ALLCACTSCLQANYTCETDG ACMVSIFNLD 61 GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDYCNRIDLRVPS GHLKEPEHPS 121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQRLDMEDPSCE MCLSKDKTLQ 181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGEVWRGRWRGGD VAVKIFSSRE 241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK DNGTWTQLWLVSDYHEHGSL FDYLNRYTVT 301 IEGMIKLALS AASGLAHLHM EIVGTQGKPG IAHRDLKSKNILVKKNGMCA IADLGLAVRH 361 DAVTDTIDIA PNQRVGTKRY MAPEVLDETI NMKHFDSFKCADIYALGLVY WEIARRCNSG 421 GVHEEYQLPY YDLVPSDPSI EEMRKVVCDQ KLRPNIPNWWQSYEALRVMG KMMRECWYAN 481 GAARLTALRI KKTLSQLSVQ EDVKI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular human ALK4 polypeptide sequence is asfollows:

(SEQ ID NO: 27) SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG PVE

A nucleic acid sequence encoding the ALK4 precursor protein is shownbelow (SEQ ID NO: 28), corresponding to nucleotides 78-1592 of GenbankReference Sequence NM_004302.4. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 28) ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGG TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAG GAAGACGTGAAGATC

A nucleic acid sequence encoding the extracellular ALK4 polypeptide isas follows:

(SEQ ID NO: 29) TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC CCGGTGGAG

An alternative isoform of human ALK4 precursor, isoform B (NCBI Ref SeqNP_064732.3), is as follows:

(SEQ ID NO: 83) 1 MVSIFNLDGM EHHVRTCIPK VELVPAGKPF YCLSSEDLRN THCCYTDYCNRIDLRVPSGH 61 LKEPEHPSMW GPVELVGIIA GPVFLLFLII IIVFLVINYH QRVYHNRQRLDMEDPSCEMC 121 LSKDKTLQDL VYDLSTSGSG SGLPLFVQRT VARTIVLQEI IGKGRFGEVWRGRWRGGDVA 181 VKIFSSREER SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVSDYHEHGSLFD 241 YLNRYTVTIE GMIKLALSAA SGLAHLHMEI VGTQGKPGIA HRDLKSKNILVKKNGMCAIA 301 DLGLAVRHDA VTDTIDIAPN QRVGTKRYMA PEVLDETINM KHFDSFKCADIYALGLVYWE 361 IARRCNSGGV HEEYQLPYYD LVPSDPSIEE MRKVVCDQKL RPNIPNWWQSYEALRVMGKM 421 MRECWYANGA ARLTALRIKK TLSQLSVQED VKI

The extracellular domain is indicated in bold font.

The extracellular ALK4 polypeptide sequence (isoform B) is as follows:

(SEQ ID NO: 84) MVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWGPVE

A nucleic acid sequence encoding isoform B of the ALK4 precursor proteinis shown below (SEQ ID NO: 85), corresponding to nucleotides 186-1547 ofGenbank Reference Sequence NM_020327.3. The extracellular domain isindicated in bold font.

(SEQ ID NO: 85) ATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAGGAAGAC GTGAAGATC

A nucleic acid sequence encoding the extracelluar domain of ALK4polypeptide (isoform B) is as follows:

(SEQ ID NO: 86) ATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK4 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK4 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK4 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK4). In other preferred embodiments, ALK4polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK4 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 26, 27, 83, 84, 125, 127, 417, or 418. In someembodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK4 polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 26, 27, 83, 84, 125, 127, 417, or 418.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK5 polypeptide. As used herein, the term “ALK5”refers to a family of activin receptor-like kinase-5 proteins from anyspecies and variants derived from such ALK4 proteins by mutagenesis orother modification. Reference to ALK5 herein is understood to be areference to any one of the currently identified forms. Members of theALK5 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK5 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK5 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK5-related polypeptides described herein is based on thenumbering of the human ALK5 precursor protein sequence below (SEQ ID NO:30), unless specifically designated otherwise.

The canonical human ALK5 precursor protein sequence (NCBI Ref SeqNP_004603.1) is as follows:

(SEQ ID NO: 30) 1 MEAAVAAPRP RLLLLVLAAA AAAA AALLPG ATALQCFCHLCTKDNFTCVT DGLCFVSVTE 61 TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYCCNQDHCNKIE LPTTVKSSPG 121 LGPVELAAVI AGPVCFVCIS LMLMVYICHN RTVIHHRVPNEEDPSLDRPF ISEGTTLKDL 181 IYDMTTSGSG SGLPLLVQRT IARTIVLQES IGKGRFGEVWRGKWRGEEVA VKIFSSREER 241 SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVSDYHEHGSLFD YLNRYTVTVE 301 GMIKLALSTA SGLAHLHMEI VGTQGKPAIA HRDLKSKNILVKKNGTCCIA DLGLAVRHDS 361 ATDTIDIAPN HRVGTKRYMA PEVLDDSINM KHFESFKRADIYAMGLVFWE IARRCSIGGI 421 HEDYQLPYYD LVPSDPSVEE MRKVVCEQKL RPNIPNRWQSCEALRVMAKI MRECWYANGA 481 ARLTALRIKK TLSQLSQQEG IKM

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK5 polypeptide sequence is as follows:

(SEQ ID NO: 31) AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPV EL

A nucleic acid sequence encoding the ALK5 precursor protein is shownbelow (SEQ ID NO: 32), corresponding to nucleotides 77-1585 of GenbankReference Sequence NM_004612.2. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 32) ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCG GCGGCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCTGCCACAACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAGGTTTACCATTGCTTGTTCAGAGAACAATTGCGAGAACTATTGTGTTACAAGAAAGCATTGGCAAAGGTCGATTTGGAGAAGTTTGGAGAGGAAAGTGGCGGGGAGAAGAAGTTGCTGTTAAGATATTCTCCTCTAGAGAAGAACGTTCGTGGTTCCGTGAGGCAGAGATTTATCAAACTGTAATGTTACGTCATGAAAACATCCTGGGATTTATAGCAGCAGACAATAAAGACAATGGTACTTGGACTCAGCTCTGGTTGGTGTCAGATTATCATGAGCATGGATCCCTTTTTGATTACTTAAACAGATACACAGTTACTGTGGAAGGAATGATAAAACTTGCTCTGTCCACGGCGAGCGGTCTTGCCCATCTTCACATGGAGATTGTTGGTACCCAAGGAAAGCCAGCCATTGCTCATAGAGATTTGAAATCAAAGAATATCTTGGTAAAGAAGAATGGAACTTGCTGTATTGCAGACTTAGGACTGGCAGTAAGACATGATTCAGCCACAGATACCATTGATATTGCTCCAAACCACAGAGTGGGAACAAAAAGGTACATGGCCCCTGAAGTTCTCGATGATTCCATAAATATGAAACATTTTGAATCCTTCAAACGTGCTGACATCTATGCAATGGGCTTAGTATTCTGGGAAATTGCTCGACGATGTTCCATTGGTGGAATTCATGAAGATTACCAACTGCCTTATTATGATCTTGTACCTTCTGACCCATCAGTTGAAGAAATGAGAAAAGTTGTTTGTGAACAGAAGTTAAGGCCAAATATCCCAAACAGATGGCAGAGCTGTGAAGCCTTGAGAGTAATGGCTAAAATTATGAGAGAATGTTGGTATGCCAATGGAGCAGCTAGGCTTACAGCATTGCGGATTAAGAAAACATTATCGCAACTCAGTCAACAGGAAGGC ATCAAAATG

A nucleic acid sequence encoding the extracellular human ALK5polypeptide is as follows:

(SEQ ID NO: 33) GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTG GAACTG

An alternative isoform of the human ALK5 precursor protein sequence,isoform 2 (NCBI Ref Seq XP_005252207.1), is as follows:

(SEQ ID NO: 87) 1 MEAAVAAPRP RLLLLVLAAA AAAA AALLPG ATALQCFCHLCTKDNFTCVT DGLCFVSVTE 61 TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYCCNQDHCNKIE LPTTGPFSVK 121 SSPGLGPVEL AAVIAGPVCF VCISLMLMVY ICHNRTVIHHRVPNEEDPSL DRPFISEGTT 181 LKDLIYDMTT SGSGSGLPLL VQRTIARTIV LQESIGKGRFGEVWRGKWRG EEVAVKIFSS 241 REERSWFREA EIYQTVMLRH ENILGFIAAD NKDNGTWTQLWLVSDYHEHG SLFDYLNRYT 301 VTVEGMIKLA LSTASGLAHL HMEIVGTQGK PAIAHRDLKSKNILVKKNGT CCIADLGLAV 361 RHDSATDTID IAPNHRVGTK RYMAPEVLDD SINMKHFESFKRADIYAMGL VFWEIARRCS 421 IGGIHEDYQL PYYDLVPSDP SVEEMRKVVC EQKLRPNIPNRWQSCEALRV MAKIMRECWY 481 ANGAARLTAL RIKKTLSQLS QQEGIKM

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK5 polypeptide sequence (isoform 2) is asfollows:

(SEQ ID NO: 88) AALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTGPFSVKSS PGLGPVEL

A nucleic acid sequence encoding human ALK5 precursor protein (isoform2) is shown below (SEQ ID NO: 89), corresponding to nucleotides 77-1597of Genbank Reference Sequence XM_0052521501 The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 89) ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCG GCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGGCCCTTTTTCAGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCTGCCACAACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAGGTTTACCATTGCTTGTTCAGAGAACAATTGCGAGAACTATTGTGTTACAAGAAAGCATTGGCAAAGGTCGATTTGGAGAAGTTTGGAGAGGAAAGTGGCGGGGAGAAGAAGTTGCTGTTAAGATATTCTCCTCTAGAGAAGAACGTTCGTGGTTCCGTGAGGCAGAGATTTATCAAACTGTAATGTTACGTCATGAAAACATCCTGGGATTTATAGCAGCAGACAATAAAGACAATGGTACTTGGACTCAGCTCTGGTTGGTGTCAGATTATCATGAGCATGGATCCCTTTTTGATTACTTAAACAGATACACAGTTACTGTGGAAGGAATGATAAAACTTGCTCTGTCCACGGCGAGCGGTCTTGCCCATCTTCACATGGAGATTGTTGGTACCCAAGGAAAGCCAGCCATTGCTCATAGAGATTTGAAATCAAAGAATATCTTGGTAAAGAAGAATGGAACTTGCTGTATTGCAGACTTAGGACTGGCAGTAAGACATGATTCAGCCACAGATACCATTGATATTGCTCCAAACCACAGAGTGGGAACAAAAAGGTACATGGCCCCTGAAGTTCTCGATGATTCCATAAATATGAAACATTTTGAATCCTTCAAACGTGCTGACATCTATGCAATGGGCTTAGTATTCTGGGAAATTGCTCGACGATGTTCCATTGGTGGAATTCATGAAGATTACCAACTGCCTTATTATGATCTTGTACCTTCTGACCCATCAGTTGAAGAAATGAGAAAAGTTGTTTGTGAACAGAAGTTAAGGCCAAATATCCCAAACAGATGGCAGAGCTGTGAAGCCTTGAGAGTAATGGCTAAAATTATGAGAGAATGTTGGTATGCCAATGGAGCAGCTAGGCTTACAGCATTGCGGATTAAGAAAACATTATCGCAACTCAGT CAACAGGAAGGCATCAAAATG

A nucleic acid sequence encoding the processed extracellular ALK5polypeptide is as follows:

(SEQ ID NO: 90) GCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGGCCCTTTTTCAGTAAAGTCATCACCTGGC CTTGGTCCTGTGGAACTG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK5 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK5 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK5 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK5). In other preferred embodiments, ALK5polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK5 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 30, 31, 87, 88, 128, 130, 419, or 420. In someembodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK5 polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 30, 31, 87, 88, 128, 130, 419, or 420.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK6 polypeptide. As used herein, the term “ALK6”refers to a family of activin receptor-like kinase-6 proteins from anyspecies and variants derived from such ALK6 proteins by mutagenesis orother modification. Reference to ALK6 herein is understood to be areference to any one of the currently identified forms. Members of theALK6 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK6 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK6 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK6-related polypeptides described herein is based on thenumbering of the human ALK6 precursor protein sequence below (SEQ ID NO:34), unless specifically designated otherwise.

The canonical human ALK6 precursor protein sequence (NCBI Ref SeqNP_001194.1) is as follows:

(SEQ ID NO: 34) 1 MLLRSAGKLN VGT KKEDGES TAPTPRPKVL RCKCHHHCPEDSVNNICSTD GYCFTMIEED 61 DSGLPVVTSG CLGLEGSDFQ CRDTPIPHQR RSIECCTERNECNKDLHPTL PPLKNRDFVD 121 GPIHHRALLI SVTVCSLLLV LIILFCYFRY KRQETRPRYSIGLEQDETYI PPGESLRDLI 181 EQSQSSGSGS GLPLLVQRTI AKQIQMVKQI GKGRYGEVWMGKWRGEKVAV KVFFTTEEAS 241 WFRETEIYQT VLMRHENILG FIAADIKGTG SWTQLYLITDYHENGSLYDY LKSTTLDAKS 301 MLKLAYSSVS GLCHLHTEIF STQGKPAIAH RDLKSKNILVKKNGTCCIAD LGLAVKFISD 361 TNEVDIPPNT RVGTKRYMPP EVLDESLNRN HFQSYIMADMYSFGLILWEV ARRCVSGGIV 421 EEYQLPYHDL VPSDPSYEDM REIVCIKKLR PSFPNRWSSDECLRQMGKLM TECWAHNPAS 481 RLTALRVKKT LAKMSESQDI KL

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK6 polypeptide sequence is as follows:

(SEQ ID NO: 35) KKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIEEDDSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPL KNRDFVDGPIHHR

A nucleic acid sequence encoding the ALK6 precursor protein is shownbelow (SEQ ID NO: 36), corresponding to nucleotides 275-1780 of GenbankReference Sequence NM_001203.2. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 36) ATGCTTTTGCGAAGTGCAGGAAAATTAAATGTGGGCACC AAGAAAGAGGATGGTGAGAGTACAGCCCCCACCCCCCGTCCAAAGGTCTTGCGTTGTAAATGCCACCACCATTGTCCAGAAGACTCAGTCAACAATATTTGCAGCACAGACGGATATTGTTTCACGATGATAGAAGAGGATGACTCTGGGTTGCCTGTGGTCACTTCTGGTTGCCTAGGACTAGAAGGCTCAGATTTTCAGTGTCGGGACACTCCCATTCCTCATCAAAGAAGATCAATTGAATGCTGCACAGAAAGGAACGAATGTAATAAAGACCTACACCCTACACTGCCTCCATTGAAAAACAGAGATTTTGTTGATGGACCTATACACCACAGGGCTTTACTTATATCTGTGACTGTCTGTAGTTTGCTCTTGGTCCTTATCATATTATTTTGTTACTTCCGGTATAAAAGACAAGAAACCAGACCTCGATACAGCATTGGGTTAGAACAGGATGAAACTTACATTCCTCCTGGAGAATCCCTGAGAGACTTAATTGAGCAGTCTCAGAGCTCAGGAAGTGGATCAGGCCTCCCTCTGCTGGTCCAAAGGACTATAGCTAAGCAGATTCAGATGGTGAAACAGATTGGAAAAGGTCGCTATGGGGAAGTTTGGATGGGAAAGTGGCGTGGCGAAAAGGTAGCTGTGAAAGTGTTCTTCACCACAGAGGAAGCCAGCTGGTTCAGAGAGACAGAAATATATCAGACAGTGTTGATGAGGCATGAAAACATTTTGGGTTTCATTGCTGCAGATATCAAAGGGACAGGGTCCTGGACCCAGTTGTACCTAATCACAGACTATCATGAAAATGGTTCCCTTTATGATTATCTGAAGTCCACCACCCTAGACGCTAAATCAATGCTGAAGTTAGCCTACTCTTCTGTCAGTGGCTTATGTCATTTACACACAGAAATCTTTAGTACTCAAGGCAAACCAGCAATTGCCCATCGAGATCTGAAAAGTAAAAACATTCTGGTGAAGAAAAATGGAACTTGCTGTATTGCTGACCTGGGCCTGGCTGTTAAATTTATTAGTGATACAAATGAAGTTGACATACCACCTAACACTCGAGTTGGCACCAAACGCTATATGCCTCCAGAAGTGTTGGACGAGAGCTTGAACAGAAATCACTTCCAGTCTTACATCATGGCTGACATGTATAGTTTTGGCCTCATCCTTTGGGAGGTTGCTAGGAGATGTGTATCAGGAGGTATAGTGGAAGAATACCAGCTTCCTTATCATGACCTAGTGCCCAGTGACCCCTCTTATGAGGACATGAGGGAGATTGTGTGCATCAAGAAGTTACGCCCCTCATTCCCAAACCGGTGGAGCAGTGATGAGTGTCTAAGGCAGATGGGAAAACTCATGACAGAATGCTGGGCTCACAATCCTGCATCAAGGCTGACAGCCCTGCGGGTTAAGAAAACACTTGCCAAAATGTCAGAGTCCCAGGACATT AAACTC

A nucleic acid sequence encoding processed extracellular ALK6polypeptide is as follows:

(SEQ ID NO: 37) AAGAAAGAGGATGGTGAGAGTACAGCCCCCACCCCCCGTCCAAAGGTCTTGCGTTGTAAATGCCACCACCATTGTCCAGAAGACTCAGTCAACAATATTTGCAGCACAGACGGATATTGTTTCACGATGATAGAAGAGGATGACTCTGGGTTGCCTGTGGTCACTTCTGGTTGCCTAGGACTAGAAGGCTCAGATTTTCAGTGTCGGGACACTCCCATTCCTCATCAAAGAAGATCAATTGAATGCTGCACAGAAAGGAACGAATGTAATAAAGACCTACACCCTACACTGCCTCCATTGAAAAACAGAGATTTTGTTGATGGACCTATACACCACAGG

An alternative isoform of human ALK6 precursor protein sequence, isoform2 (NCBI Ref Seq NP_001243722.1) is as follows:

(SEQ ID NO: 91) 1 MGWLEELNWQ LHIFLLILLS MHTRA NFLDN MLLRSAGKLNVGTKKEDGES TAPTPRPKVL 61 RCKCHHHCPE DSVNNICSTD GYCFTMIEED DSGLPVVTSGCLGLEGSDFQ CRDTPIPHQR 121 RSIECCTERN ECNKDLHPTL PPLKNRDFVD GPIHHRALLISVTVCSLLLV LIILFCYFRY 181 KRQETRPRYS IGLEQDETYI PPGESLRDLI EQSQSSGSGSGLPLLVQRTI AKQIQMVKQI 241 GKGRYGEVWM GKWRGEKVAV KVFFTTEEAS WFRETEIYQTVLMRHENILG FIAADIKGTG 301 SWTQLYLITD YHENGSLYDY LKSTTLDAKS MLKLAYSSVSGLCHLHTEIF STQGKPAIAH 361 RDLKSKNILV KKNGTCCIAD LGLAVKFISD TNEVDIPPNTRVGTKRYMPP EVLDESLNRN 421 HFQSYIMADM YSFGLILWEV ARRCVSGGIV EEYQLPYHDLVPSDPSYEDM REIVCIKKLR 481 PSFPNRWSSD ECLRQMGKLM TECWAHNPAS RLTALRVKKTLAKMSESQDI KL

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK6 polypeptide sequence (isoform 2) is asfollows:

(SEQ ID NO: 92) NFLDNMLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIEEDDSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPLKNRDFVDGPIHHR

A nucleic acid sequence encoding human ALK6 precursor protein (isoform2) is shown below, corresponding to nucleotides 22-1617 of GenbankReference Sequence NM_001256793.1. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 93) ATGGGTTGGCTGGAAGAACTAAACTGGCAGCTTCACATTTTCTTGCTCATTCTTCTCTCTATGCACACAAGGGCA AACTTCCTTGATAACATGCTTTTGCGAAGTGCAGGAAAATTAAATGTGGGCACCAAGAAAGAGGATGGTGAGAGTACAGCCCCCACCCCCCGTCCAAAGGTCTTGCGTTGTAAATGCCACCACCATTGTCCAGAAGACTCAGTCAACAATATTTGCAGCACAGACGGATATTGTTTCACGATGATAGAAGAGGATGACTCTGGGTTGCCTGTGGTCACTTCTGGTTGCCTAGGACTAGAAGGCTCAGATTTTCAGTGTCGGGACACTCCCATTCCTCATCAAAGAAGATCAATTGAATGCTGCACAGAAAGGAACGAATGTAATAAAGACCTACACCCTACACTGCCTCCATTGAAAAACAGAGATTTTGTTGATGGACCTATACACCACAGGGCTTTACTTATATCTGTGACTGTCTGTAGTTTGCTCTTGGTCCTTATCATATTATTTTGTTACTTCCGGTATAAAAGACAAGAAACCAGACCTCGATACAGCATTGGGTTAGAACAGGATGAAACTTACATTCCTCCTGGAGAATCCCTGAGAGACTTAATTGAGCAGTCTCAGAGCTCAGGAAGTGGATCAGGCCTCCCTCTGCTGGTCCAAAGGACTATAGCTAAGCAGATTCAGATGGTGAAACAGATTGGAAAAGGTCGCTATGGGGAAGTTTGGATGGGAAAGTGGCGTGGCGAAAAGGTAGCTGTGAAAGTGTTCTTCACCACAGAGGAAGCCAGCTGGTTCAGAGAGACAGAAATATATCAGACAGTGTTGATGAGGCATGAAAACATTTTGGGTTTCATTGCTGCAGATATCAAAGGGACAGGGTCCTGGACCCAGTTGTACCTAATCACAGACTATCATGAAAATGGTTCCCTTTATGATTATCTGAAGTCCACCACCCTAGACGCTAAATCAATGCTGAAGTTAGCCTACTCTTCTGTCAGTGGCTTATGTCATTTACACACAGAAATCTTTAGTACTCAAGGCAAACCAGCAATTGCCCATCGAGATCTGAAAAGTAAAAACATTCTGGTGAAGAAAAATGGAACTTGCTGTATTGCTGACCTGGGCCTGGCTGTTAAATTTATTAGTGATACAAATGAAGTTGACATACCACCTAACACTCGAGTTGGCACCAAACGCTATATGCCTCCAGAAGTGTTGGACGAGAGCTTGAACAGAAATCACTTCCAGTCTTACATCATGGCTGACATGTATAGTTTTGGCCTCATCCTTTGGGAGGTTGCTAGGAGATGTGTATCAGGAGGTATAGTGGAAGAATACCAGCTTCCTTATCATGACCTAGTGCCCAGTGACCCCTCTTATGAGGACATGAGGGAGATTGTGTGCATCAAGAAGTTACGCCCCTCATTCCCAAACCGGTGGAGCAGTGATGAGTGTCTAAGGCAGATGGGAAAACTCATGACAGAATGCTGGGCTCACAATCCTGCATCAAGGCTGACAGCCCTGCGGGTTAAGAAAACACTTGCCAAAATGTCAGAGTCCCAGGACATTAAACTC

A nucleic acid sequence encoding the processed extracellular ALK6polypeptide is as follows:

(SEQ ID NO: 94) AACTTCCTTGATAACATGCTTTTGCGAAGTGCAGGAAAATTAAATGTGGGCACCAAGAAAGAGGATGGTGAGAGTACAGCCCCCACCCCCCGTCCAAAGGTCTTGCGTTGTAAATGCCACCACCATTGTCCAGAAGACTCAGTCAACAATATTTGCAGCACAGACGGATATTGTTTCACGATGATAGAAGAGGATGACTCTGGGTTGCCTGTGGTCACTTCTGGTTGCCTAGGACTAGAAGGCTCAGATTTTCAGTGTCGGGACACTCCCATTCCTCATCAAAGAAGATCAATTGAATGCTGCACAGAAAGGAACGAATGTAATAAAGACCTACACCCTACACTGCCTCCATTGAAAAACAGAGATTTTGTTGATGGACCTATACACCACAGG

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK6 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK6 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK6 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK6). In other preferred embodiments, ALK6polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK6 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 34, 35, 91, 92, 131, 133, 421, or 422. In someembodiments, single-arm heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK6 polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 34, 35, 91, 92, 131, 133, 421, or 422.

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK7 polypeptide. As used herein, the term “ALK7”refers to a family of activin receptor-like kinase-7 proteins from anyspecies and variants derived from such ALK7 proteins by mutagenesis orother modification. Reference to ALK7 herein is understood to be areference to any one of the currently identified forms. Members of theALK7 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK7 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK7 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK7-related polypeptides described herein is based on thenumbering of the human ALK7 precursor protein sequence below (SEQ ID NO:38), unless specifically designated otherwise.

Several naturally occurring isoforms of human ALK7 have been described.The sequence of canonical human ALK7 isoform 1 precursor protein (NCBIRef Seq NP_660302.2) is as follows:

(SEQ ID NO: 38) 1 MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTC QTEGACWASVMLTNGKEQVI 61 KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP TASPNAPKLGPMELAIIITV 121 PVCLLSIAAM LTVWACQGRQ CSYRKKKRPN VEEPLSECNL VNAGKTLKDLIYDVTASGSG 181 SGLPLLVQRT IARTIVLQEI VGKGRFGEVW HGRWCGEDVA VKIFSSRDERSWFREAEIYQ 241 TVMLRHENIL GFIAADNKDN GTWTQLWLVS EYHEQGSLYD YLNRNIVTVAGMIKLALSIA 301 SGLAHLHMEI VGTQGKPAIA HRDIKSKNIL VKKCETCAIA DLGLAVKHDSILNTIDIPQN 361 PKVGTKRYMA PEMLDDTMNV NIFESFKRAD IYSVGLVYWE IARRCSVGGIVEEYQLPYYD 421 MVPSDPSIEE MRKVVCDQKF RPSIPNQWQS CEALRVMGRI MRECWYANGAARLTALRIKK 481 TISQLCVKED CKA

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK7 isoform 1 polypeptide sequence is asfollows:

(SEQ ID NO: 39) ELSPGLKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME

A nucleic acid sequence encoding human ALK7 isoform 1 precursor proteinis shown below (SEQ ID NO: 40), corresponding to nucleotides 244-1722 ofGenbank Reference Sequence NM_145259.2. The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 40) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCC GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCATCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGACAGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACCAAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCTGAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

A nucleic acid sequence encoding the processed extracellular ALK7polypeptide (isoform 1) is as follows:

(SEQ ID NO: 41) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAG

The amino acid sequence of an alternative isoform of human ALK7, isoform2 (NCBI Ref Seq NP_001104501.1), is shown in its processed form asfollows (SEQ ID NO: 301), where the extracellular domain is indicated inbold font.

(SEQ ID NO: 301) 1 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTDFCNNITLHLP TASPNAPKLG 61 PMELAIIITV PVCLLSIAAM LTVWACQGRQ CSYRKKKRPNVEEPLSECNL VNAGKTLKDL 121 IYDVTASGSG SGLPLLVQRT IARTIVLQEI VGKGRFGEVWHGRWCGEDVA VKIFSSRDER 181 SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVSEYHEQGSLYD YLNRNIVTVA 241 GMIKLALSIA SGLAHLHMEI VGTQGKPAIA HRDIKSKNILVKKCETCAIA DLGLAVKHDS 301 ILNTIDIPQN PKVGTKRYMA PEMLDDTMNV NIFESFKRADIYSVGLVYWE IARRCSVGGI 361 VEEYQLPYYD MVPSDPSIEE MRKVVCDQKF RPSIPNQWQSCEALRVMGRI MRECWYANGA 421 ARLTALRIKK TISQLCVKED CKA

The amino acid sequence of the extracellular ALK7 polypeptide (isoform2) is as follows:

(SEQ ID NO: 302) MLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME.

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform2) is shown below (SEQ ID NO: 303), corresponding to nucleotides279-1607 of NCBI Reference Sequence NM_001111031.1. The extracellulardomain is indicated in bold font.

(SEQ ID NO: 303) ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCATCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGACAGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACCAAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCTGAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

A nucleic acid sequence encoding the extracellular ALK7 polypeptide(isoform 2) is as follows (SEQ ID NO: 304):

(SEQ ID NO: 304) ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAG

The amino acid sequence of an alternative human ALK7 precursor protein,isoform 3 (NCBI Ref Seq NP_001104502.1), is shown as follows (SEQ ID NO:305), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 305) 1 MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTCQTEGACWASV MLTNGKEQVI 61 KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLPTGLPLLVQRT IARTIVLQEI 121 VGKGRFGEVW HGRWCGEDVA VKIFSSRDER SWFREAEIYQTVMLRHENIL GFIAADNKDN 181 GTWTQLWLVS EYHEQGSLYD YLNRNIVTVA GMIKLALSIASGLAHLHMEI VGTQGKPAIA 241 HRDIKSKNIL VKKCETCAIA DLGLAVKHDS ILNTIDIPQNPKVGTKRYMA PEMLDDTMNV 301 NIFESFKRAD IYSVGLVYWE IARRCSVGGI VEEYQLPYYDMVPSDPSIEE MRKVVCDQKF 361 RPSIPNQWQS CEALRVMGRI MRECWYANGA ARLTALRIKKTISQLCVKED CKA

The amino acid sequence of the processed ALK7 polypeptide (isoform 3) isas follows (SEQ ID NO: 306). This isoform lacks a transmembrane domainand is therefore proposed to be soluble in its entirety (Roberts et al.,2003, Biol Reprod 68:1719-1726). N-terminal variants of SEQ ID NO: 306are predicted as explained below.

(SEQ ID NO: 306) 1 ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVIKSCVSLPELN AQVFCHSSNN 61 VTKTECCFTD FCNNITLHLP TGLPLLVQRT IARTIVLQEIVGKGRFGEVW HGRWCGEDVA 121 VKIFSSRDER SWFREAEIYQ TVMLRHENIL GFIAADNKDNGTWTQLWLVS EYHEQGSLYD 181 YLNRNIVTVA GMIKLALSIA SGLAHLHMEI VGTQGKPAIAHRDIKSKNIL VKKCETCAIA 241 DLGLAVKHDS ILNTIDIPQN PKVGTKRYMA PEMLDDTMNVNIFESFKRAD IYSVGLVYWE 301 IARRCSVGGI VEEYQLPYYD MVPSDPSIEE MRKVVCDQKFRPSIPNQWQS CEALRVMGRI 361 MRECWYANGA ARLTALRIKK TISQLCVKED CKA

A nucleic acid sequence encoding the unprocessed ALK7 polypeptideprecursor protein (isoform 3) is shown below (SEQ ID NO: 307),corresponding to nucleotides 244-1482 of NCBI Reference SequenceNM_001111032.1. The signal sequence is indicated by solid underline.

(SEQ ID NO: 307) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform3) is as follows (SEQ ID NO: 308):

(SEQ ID NO: 308) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

The amino acid sequence of an alternative human ALK7 precursor protein,isoform 4 (NCBI Ref Seq NP_001104503.1), is shown as follows (SEQ ID NO:309), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 309) 1 MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTCQTEGACWASV MLTNGKEQVI 61 KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLPTDNGTWTQLW LVSEYHEQGS 121 LYDYLNRNIV TVAGMIKLAL SIASGLAHLH MEIVGTQGKPAIAHRDIKSK NILVKKCETC 181 AIADLGLAVK HDSILNTIDI PQNPKVGTKR YMAPEMLDDTMNVNIFESFK RADIYSVGLV 241 YWEIARRCSV GGIVEEYQLP YYDMVPSDPS IEEMRKVVCDQKFRPSIPNQ WQSCEALRVM 301 GRIMRECWYA NGAARLTALR IKKTISQLCV KEDCKA

The amino acid sequence of the processed ALK7 polypeptide (isoform 4) isas follows (SEQ ID NO: 310). Like ALK7 isoform 3, isoform 4 lacks atransmembrane domain and is therefore proposed to be soluble in itsentirety (Roberts et al., 2003, Biol Reprod 68:1719-1726). N-terminalvariants of SEQ ID NO: 310 are predicted as explained below.

(SEQ ID NO: 310) 1 ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVIKSCVSLPELN AQVFCHSSNN 61 VTKTECCFTD FCNNITLHLP TDNGTWTQLW LVSEYHEQGSLYDYLNRNIV TVAGMIKLAL 121 SIASGLAHLH MEIVGTQGKP AIAHRDIKSK NILVKKCETCAIADLGLAVK HDSILNTIDI 181 PQNPKVGTKR YMAPEMLDDT MNVNIFESFK RADIYSVGLVYWEIARRCSV GGIVEEYQLP 240 YYDMVPSDPS IEEMRKVVCD QKFRPSIPNQ WQSCEALRVMGRIMRECWYA NGAARLTALR 301 IKKTISQLCV KEDCKA

A nucleic acid sequence encoding the unprocessed ALK7 polypeptideprecursor protein (isoform 4) is shown below (SEQ ID NO: 311),corresponding to nucleotides 244-1244 of NCBI Reference SequenceNM_001111033.1. The signal sequence is indicated by solid underline.

(SEQ ID NO: 311) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACT GCAAAGCCTAA

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform4) is as follows (SEQ ID NO: 312):

(SEQ ID NO: 312) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC TAA

Based on the signal sequence of full-length ALK7 (isoform 1) in the rat(see NCBI Reference Sequence NP_620790.1) and on the high degree ofsequence identity between human and rat ALK7, it is predicted that aprocessed form of human ALK7 isoform 1 is as follows (SEQ ID NO: 313).

(SEQ ID NO: 313) 1 LKCVCLLCDS SNFTCQTEGA CWASVMLTNG KEQVIKSCVSLPELNAQVFC HSSNNVTKTE 61 CCFTDFCNNI TLHLPTASPN APKLGPME

Active variants of processed ALK7 isoform 1 are predicted in which SEQID NO: 39 is truncated by 1, 2, 3, 4, 5, 6, or 7 amino acids at theN-terminus and SEQ ID NO: 313 is truncated by 1 or 2 amino acids at theN-terminus. Consistent with SEQ ID NO: 313, it is further expected thatleucine is the N-terminal amino acid in the processed forms of humanALK7 isoform 3 (SEQ ID NO: 306) and human ALK7 isoform 4 (SEQ ID NO:310).

In certain embodiments, the disclosure relates to single-armheteromultimer complexes that comprise at least one ALK7 polypeptide,which includes fragments, functional variants, and modified formsthereof. Preferably, ALK7 polypeptides for use in accordance withinventions of the disclosure (e.g., single-arm heteromultimer complexescomprising an ALK7 polypeptide and uses thereof) are soluble (e.g., anextracellular domain of ALK7). In other preferred embodiments, ALK7polypeptides for use in accordance with the inventions of the disclosurebind to and/or inhibit (antagonize) activity (e.g., induction of Smad2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, single-arm heteromultimer complexes of thedisclosure comprise at least one ALK7 polypeptide that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 38, 39, 134, 136, 301, 302, 305, 306, 309, 310,313, 423, or 424. In some embodiments, single-arm heteromultimercomplexes of the disclosure consist or consist essentially of at leastone ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO: 38, 39,134, 136, 301, 302, 305, 306, 309, 310, 313, 423, or 424.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of a TGF-beta superfamilytype I receptor polypeptide (e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6,and ALK7) or a TGF-beta superfamily type II receptor polypeptide (e.g.,ActRIIA, ActRIIB, TGFBRII, BMPRII, and MISRII) for such purposes asenhancing therapeutic efficacy or stability (e.g., shelf-life andresistance to proteolytic degradation in vivo). Variants can be producedby amino acid substitution, deletion, addition, or combinations thereof.For instance, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (e.g., conservative mutations) willnot have a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Whether achange in the amino acid sequence of a polypeptide of the disclosureresults in a functional homolog can be readily determined by assessingthe ability of the variant polypeptide to produce a response in cells ina fashion similar to the wild-type polypeptide, or to bind to one ormore TGF-beta ligands including, for example, BMP2, BMP2/7, BMP3, BMP4,BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, nodal, GDNF, neurturin, artemin, persephin, MIS, and Lefty.

In certain embodiments, the present disclosure contemplates specificmutations of a TGF-beta superfamily type I receptor polypeptide (e.g.,ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7) or a TGF-beta superfamilytype II receptor polypeptide (e.g., ActRIIA, ActRIIB, TGFBRII, BMPRII,and MISRII) of the disclosure so as to alter the glycosylation of thepolypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine orasparagine-X-serine (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically. Chemical deglycosylationmay involve, for example, exposure of a polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987)138:350]. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect, and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, TGF-beta superfamily type I and II receptor single-armcomplexes of the present disclosure for use in humans may be expressedin a mammalian cell line that provides proper glycosylation, such asHEK293 or CHO cell lines, although other mammalian expression cell linesare expected to be useful as well.

The present disclosure further contemplates a method of generatingmutants, particularly sets of combinatorial mutants of a TGF-betasuperfamily type I receptor polypeptide (e.g., ALK1, ALK2, ALK3, ALK4,ALK5, ALK6, and ALK7) or a TGF-beta superfamily type II receptorpolypeptide (e.g., ActRIIA, ActRIIB, TGFBRII, BMPRII, and MISRII) of thepresent disclosure, as well as truncation mutants. Pools ofcombinatorial mutants are especially useful for identifying TGF-betasuperfamily type I or TGF-beta superfamily type II receptor sequences.The purpose of screening such combinatorial libraries may be togenerate, for example, polypeptides variants which have alteredproperties, such as altered pharmacokinetic or altered ligand binding. Avariety of screening assays are provided below, and such assays may beused to evaluate variants. For example, TGF-beta superfamily type I ortype II receptor polypeptide variants may be screened for ability tobind to a TGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3, BMP4,BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, activin AE, activin BC, activin BE, nodal, GDNF, neurturin,artemin, persephin, MIS, and Lefty), to prevent binding of a TGF-betasuperfamily ligand to a TGF-beta superfamily receptor, and/or tointerfere with signaling caused by an TGF-beta superfamily ligand.

The activity of a TGF-beta superfamily receptor single-armheteromultimer complex of the disclosure also may be tested in acell-based or in vivo assay. For example, the effect of a single-armheteromultimer complex on the expression of genes involved in muscleproduction in a muscle cell may be assessed. This may, as needed, beperformed in the presence of one or more recombinant TGF-betasuperfamily ligand proteins (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13,GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2,TGF-β3, activin A, activin B, activin C, activin E, activin AB, activinAC, activin AE, activin BC, activin BE, nodal, GDNF, neurturin, artemin,persephin, MIS, and Lefty), and cells may be transfected so as toproduce a TGF-beta superfamily type I or type II receptor single-armcomplex, and optionally, a TGF-beta superfamily ligand. Likewise, asingle-arm heteromultimer complex of the disclosure may be administeredto a mouse or other animal, and one or more measurements, such as muscleformation and strength may be assessed using art-recognized methods.Similarly, the activity of a TGF-beta superfamily receptor polypeptideor its variants may be tested in osteoblasts, adipocytes, and/orneuronal cells for any effect on growth of these cells, for example, bythe assays as described herein and those of common knowledge in the art.A SMAD-responsive reporter gene may be used in such cell lines tomonitor effects on downstream signaling.

Combinatorial-derived variants can be generated which have increasedselectivity or generally increased potency relative to a referenceTGF-beta superfamily receptor single-arm heteromultimer complex. Suchvariants, when expressed from recombinant DNA constructs, can be used ingene therapy protocols. Likewise, mutagenesis can give rise to variantswhich have extracellular half-lives dramatically different than thecorresponding unmodified TGF-beta superfamily receptor single-armheteromultimer complex. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or othercellular processes which result in destruction, or otherwiseinactivation, of an unmodified polypeptide. Such variants, and the geneswhich encode them, can be utilized to alter polypeptide complex levelsby modulating the half-life of the polypeptide. For instance, a shorthalf-life can give rise to more transient biological effects and, whenpart of an inducible expression system, can allow tighter control ofrecombinant polypeptide complex levels outside the cell. In an Fc fusionprotein, mutations may be made in the linker (if any) and/or the Fcportion to alter the half-life of the TGF-beta superfamily receptorsingle-arm heteromultimer complex.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential TGF-beta superfamily type I or type II receptorsequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential TGF-beta superfamily type I or type II receptor encodingnucleotide sequences are expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art. See, e.g., Narang, S A (1983)Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins. See, e.g., Scott et al., (1990) Science 249:386-390; Robertset al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249:404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815.

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure can be generatedand isolated from a library by screening using, for example, alaninescanning mutagenesis [see, e.g., Ruf et al. (1994) Biochemistry33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint etal. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem.218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892;Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al.(1989) Science 244:1081-1085], by linker scanning mutagenesis [see,e.g., Gustin et al. (1993) Virology 193:653-660; and Brown et al. (1992)Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316],by saturation mutagenesis [see, e.g., Meyers et al., (1986) Science232:613]; by PCR mutagenesis [see, e.g., Leung et al. (1989) Method CellMol Biol 1:11-19]; or by random mutagenesis, including chemicalmutagenesis [see, e.g., Miller et al. (1992) A Short Course in BacterialGenetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al.(1994) Strategies in Mol Biol 7:32-34]. Linker scanning mutagenesis,particularly in a combinatorial setting, is an attractive method foridentifying truncated (bioactive) forms of TGF-beta superfamily type Ior type II receptor polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure. The most widely usedtechniques for screening large gene libraries typically comprise cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Preferred assays include bindingassays and/or cell-signaling assays for TGF-beta superfamily ligands(e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b,BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15,GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B,activin C, activin E, activin AB, activin AC, activin AE, activin BC,activin BE, nodal, GDNF, neurturin, artemin, persephin, MIS, and Lefty).

In certain embodiments, TGF-beta superfamily type I and type II receptorsingle-arm heteromultimer complexes of the disclosure may furthercomprise post-translational modifications in addition to any that arenaturally present in the TGF-beta superfamily type I or type II receptorpolypeptide. Such modifications include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. As a result, the TGF-beta superfamily type I or type IIreceptor single-arm heteromultimer complex may comprise non-amino acidelements, such as polyethylene glycols, lipids, polysaccharide ormonosaccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a single-arm heteromultimer complex may betested as described herein for other single-arm heteromultimer complexvariants. When a polypeptide of the disclosure is produced in cells bycleaving a nascent form of the polypeptide, post-translationalprocessing may also be important for correct folding and/or function ofthe protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3or HEK293) have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the TGF-betasuperfamily type I or type II receptor polypeptide.

In certain aspects, the polypeptides disclosed herein may form proteincomplexes comprising at least one TGF-beta superfamily type I or type IIreceptor polypeptide associated, covalently or non-covalently, with atleast one polypeptide comprising a complementary member of aninteraction pair. Preferably, polypeptides disclosed herein formsingle-arm heterodimeric complexes, although higher orderheteromultimeric complexes (heteromultimers) are also included such as,but not limited to, heterotrimers, heterotetramers, and furtheroligomeric structures (see, e.g., FIG. 1). In some embodiments, TGF-betasuperfamily type I or type II receptor polypeptides of the presentdisclosure comprise at least one multimerization domain. As disclosedherein, the term “multimerization domain” refers to an amino acid orsequence of amino acids that promote covalent or non-covalentinteraction between at least a first polypeptide and at least a secondpolypeptide. Polypeptides disclosed herein may be joined covalently ornon-covalently to a multimerization domain. Preferably, amultimerization domain promotes interaction between a single-armpolypeptide (e.g., a fusion polypeptide comprising a TGF-betasuperfamily type I receptor polypeptide or TGF-beta superfamily type IIreceptor polypeptide) and a complementary member of an interaction pairto promote heteromultimer formation (e.g., heterodimer formation), andoptionally hinders or otherwise disfavors homomultimer formation (e.g.,homodimer formation), thereby increasing the yield of desiredheteromultimer (see, e.g., FIG. 2).

Many methods known in the art can be used to generate TGF-betasuperfamily receptor single-arm complexes of the disclosure. Forexample, non-naturally occurring disulfide bonds may be constructed byreplacing on a first polypeptide (e.g., a fusion polypeptide comprisinga TGF-beta superfamily type I or type II receptor polypeptide) anaturally occurring amino acid with a free thiol-containing residue,such as cysteine, such that the free thiol interacts with another freethiol-containing residue on a second polypeptide (e.g., a complementarymember of an interaction pair) such that a disulfide bond is formedbetween the first and second polypeptides. Additional examples ofinteractions to promote heteromultimer formation include, but are notlimited to, ionic interactions such as described in Kjaergaard et al.,WO2007147901; electrostatic steering effects such as described in Kannanet al., U.S. Pat. No. 8,592,562; coiled-coil interactions such asdescribed in Christensen et al., U.S. 20120302737; leucine zippers suchas described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584;and helix-turn-helix motifs such as described in Pack et al., (1993)Bio/Technology 11: 1271-1277. Linkage of the various segments may beobtained via, e.g., covalent binding such as by chemical cross-linking,peptide linkers, disulfide bridges, etc., or affinity interactions suchas by avidin-biotin or leucine zipper technology.

In certain aspects, a multimerization domain may comprise one componentof an interaction pair. In some embodiments, the polypeptides disclosedherein may form protein complexes comprising a first polypeptidecovalently or non-covalently associated with a second polypeptide,wherein the first polypeptide comprises the amino acid sequence of aTGF-beta superfamily type I or type II receptor polypeptide and theamino acid sequence of a first member of an interaction pair; and thesecond polypeptide comprises the amino acid sequence of a second memberof an interaction pair. The interaction pair may be any two polypeptidesequences that interact to form a complex, particularly a heterodimericcomplex although operative embodiments may also employ an interactionpair that can form a homodimeric complex. One member of the interactionpair may be fused to a TGF-beta superfamily type I or type II receptorpolypeptide as described herein, including for example, a polypeptidesequence comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence of any one of SEQ ID NOs: 2, 3,5, 6, 10, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 68, 72, 76, 80,84, 88, 92, 302, 306, 310, and 313. An interaction pair may be selectedto confer an improved property/activity such as increased serumhalf-life, or to act as an adaptor on to which another moiety isattached to provide an improved property/activity. For example, apolyethylene glycol moiety may be attached to one or both components ofan interaction pair to provide an improved property/activity such asimproved serum half-life.

The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate. Accordingly, firstand second members of an asymmetric interaction pair may associate toform a heterodimeric interaction-pair complex (see, e.g., FIG. 2).Alternatively, the interaction pair may be unguided, meaning that themembers of the pair may associate with each other or self-associatewithout substantial preference and thus may have the same or differentamino acid sequences. Accordingly, first and second members of anunguided interaction pair may associate to form a homodimerinteraction-pair complex or a heterodimeric action-pair complex.Optionally, the first member of the interaction pair (e.g., anasymmetric pair or an unguided interaction pair) associates covalentlywith the second member of the interaction pair. Optionally, the firstmember of the interaction pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates non-covalently with the second member ofthe interaction pair.

As specific examples, the present disclosure provides fusion proteincomplexes comprising at least one TGF-beta superfamily type I or type IIreceptor polypeptide fused to a polypeptide comprising a constant domainof an immunoglobulin, such as a CH1, CH2, or CH3 domain of animmunoglobulin or an Fc domain. Fc domains derived from human IgG1,IgG2, IgG3, and IgG4 are provided herein. Other mutations are known thatdecrease either CDC or ADCC activity, and collectively, any of thesevariants are included in the disclosure and may be used as advantageouscomponents of a single-arm heteromultimeric complex of the disclosure.Optionally, the IgG1 Fc domain of SEQ ID NO: 208 has one or moremutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered inaccordance with the corresponding full-length IgG1). In certain cases,the mutant Fc domain having one or more of these mutations (e.g.,Asp-265 mutation) has reduced ability of binding to the Fcγ receptorrelative to a wildtype Fc domain. In other cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asn-434 mutation) hasincreased ability of binding to the MEW class I-related Fc-receptor(FcRN) relative to a wildtype Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 208). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising amino acid sequences with 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 208. Naturallyoccurring variants in G1Fc would include E134D and M136L according tothe numbering system used in SEQ ID NO: 208 (see Uniprot P01857).

(SEQ ID NO: 208) 1 

51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD  WLNGKEYKCK 101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ  VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV  DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 209). Dottedunderline indicates the hinge region and double underline indicatespositions where there are database conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising amino acid sequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% identity to SEQ ID NO: 209.

(SEQ ID NO: 209) 1

51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL  NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS  LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK  SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 210) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 211) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptides comprisingamino acid sequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NOs: 210 and 211.

(SEQ ID NO: 210) 1 

51  VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV  LTVLHQDWLN 101 GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR  EEMTKNQVSL 151 TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF  LYSKLTVDKS 201 RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK (SEQ ID NO: 211) 1

51 

101  EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV  LHQDWLNGKE 151 YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM  TKNQVSLTCL 201 VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS  KLTVDKSRWQ 251 QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169de1,F221Y when converted to the numbering system used in SEQ ID NO: 210, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 212). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising amino acid sequences with 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identity to SEQ ID NO: 212.

(SEQ ID NO: 212) 1

51 EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV  LHQDWLNGKE 101YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM  TKNQVSLTCL 151VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS  RLTVDKSRWQ 201EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 208), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 5. Due to unequal hinge lengths, analogous Fcpositions based on isotype alignment (FIG. 5) possess different aminoacid numbers in SEQ ID NOs: 208, 209, 210, and 212. It can also beappreciated that a given amino acid position in an immunoglobulinsequence consisting of hinge, C_(H)2, and C_(H)3 regions (e.g., SEQ IDNOs: 208, 209, 210, 211, or 212) will be identified by a differentnumber than the same position when numbering encompasses the entire IgG1heavy-chain constant domain (consisting of the C_(H)1, hinge, C_(H)2,and C_(H)3 regions) as in the Uniprot database. For example,correspondence between selected C_(H)3 positions in a human G1Fcsequence (SEQ ID NO: 208), the human IgG1 heavy chain constant domain(Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of C_(H)3 Positions in Different Numbering Systems G1Fc(Numbering IgG1 heavy chain IgG1 heavy chain begins at first constantdomain (EU numbering threonine in (Numbering scheme of Kabat hingeregion) begins at C_(H)1) et al., 1991*) Y127 Y232 Y349 S132 S237 S354E134 E239 E356 T144 T249 T366 L146 L251 L368 K170 K275 K392 D177 D282D399 Y185 Y290 Y407 K187 K292 K409 *Kabat et al. (eds) 1991; pp. 688-696in Sequences of Proteins of Immunological Interest, 5^(th) ed., Vol. 1,NIH, Bethesda, MD.

A problem that arises in large-scale production of asymmetricimmunoglobulin-based proteins from a single cell line is known as the“chain association issue”. As confronted prominently in the productionof bispecific antibodies, the chain association issue concerns thechallenge of efficiently producing a desired multichain protein fromamong the multiple combinations that inherently result when differentheavy chains and/or light chains are produced in a single cell line[see, for example, Klein et al (2012) mAbs 4:653-663]. This problem ismost acute when two different heavy chains and two different lightchains are produced in the same cell, in which case there are a total of16 possible chain combinations (although some of these are identical)when only one is typically desired. Nevertheless, the same principleaccounts for diminished yield of a desired multichain fusion proteinthat incorporates only two different (asymmetric) heavy chains.

Various methods are known in the art that increase desired pairing ofFc-containing fusion polypeptide chains in a single cell line to producea preferred asymmetric fusion protein at acceptable yields [see, forexample, Klein et al (2012) mAbs 4:653-663]. Methods to obtain desiredpairing of Fc-containing chains include, but are not limited to,charge-based pairing (electrostatic steering), “knobs-into-holes” stericpairing, SEEDbody pairing, and leucine zipper-based pairing. See, forexample, Ridgway et al (1996) Protein Eng 9:617-621; Merchant et al(1998) Nat Biotech 16:677-681; Davis et al (2010) Protein Eng Des Sel23:195-202; Gunasekaran et al (2010); 285:19637-19646; Wranik et al(2012) J Biol Chem 287:43331-43339; U.S. Pat. No. 5,932,448; WO1993/011162; WO 2009/089004, and WO 2011/034605.

For example, one means by which interaction between specificpolypeptides may be promoted is by engineering protuberance-into-cavity(knob-into-holes) complementary regions such as described in Arathoon etal., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide (e.g., a firstinteraction pair) with larger side chains (e.g., tyrosine ortryptophan). Complementary “cavities” of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide (e.g., a second interaction pair) by replacing large aminoacid side chains with smaller ones (e.g., alanine or threonine). Where asuitably positioned and dimensioned protuberance or cavity exists at theinterface of either the first or second polypeptide, it is onlynecessary to engineer a corresponding cavity or protuberance,respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negativelycharged and lysine, arginine, and histidine are positively charged.These charged residues can be used to promote heterodimer formation andat the same time hinder homodimer formation. Attractive interactionstake place between opposite charges and repulsive interactions occurbetween like charges. In part, protein complexes disclosed herein makeuse of the attractive interactions for promoting heteromultimerformation (e.g., heterodimer formation), and optionally repulsiveinteractions for hindering homodimer formation (e.g., homodimerformation) by carrying out site directed mutagenesis of chargedinterface residues.

For example, the IgG1 CH3 domain interface comprises four unique chargeresidue pairs involved in domain-domain interactions: Asp356-Lys439′,Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering inthe second chain is indicated by (′)]. It should be noted that thenumbering scheme used here to designate residues in the IgG1 CH3 domainconforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetrypresent in the CH3-CH3 domain interactions, each unique interaction willrepresented twice in the structure (e.g., Asp-399-Lys409′ andLys409-Asp399′). In the wild-type sequence, K409-D399′ favors bothheterodimer and homodimer formation. A single mutation switching thecharge polarity (e.g., K409E; positive to negative charge) in the firstchain leads to unfavorable interactions for the formation of the firstchain homodimer. The unfavorable interactions arise due to the repulsiveinteractions occurring between the same charges (negative-negative;K409E-D399′ and D399-K409E′). A similar mutation switching the chargepolarity (D399K′; negative to positive) in the second chain leads tounfavorable interactions (K409′-D399K′ and D399K-K409′) for the secondchain homodimer formation. But, at the same time, these two mutations(K409E and D399K′) lead to favorable interactions (K409E-D399K′ andD399-K409′) for the heterodimer formation.

The electrostatic steering effect on heterodimer formation and homodimerdiscouragement can be further enhanced by mutation of additional chargeresidues which may or may not be paired with an oppositely chargedresidue in the second chain including, for example, Arg355 and Lys360.The table below lists possible charge change mutations that can be used,alone or in combination, to enhance heteromultimer formation of thepolypeptide complexes disclosed herein.

Examples of Pair-Wise Charged Residue Mutations to Enhance HeterodimerFormation Interacting Corresponding Position in Mutation in position inmutation in first chain first chain second chain second chain Lys409 Aspor Glu Asp399′ Lys, Arg, or His Lys392 Asp or Glu Asp399′ Lys, Arg, orHis Lys439 Asp or Glu Asp356′ Lys, Arg, or His Lys370 Asp or Glu Glu357′Lys, Arg, or His Asp399 Lys, Arg, or His Lys409′ Asp or Glu Asp399 Lys,Arg, or His Lys392′ Asp or Glu Asp356 Lys, Arg, or His Lys439′ Asp orGlu Glu357 Lys, Arg, or His Lys370′ Asp or Glu

In some embodiments, one or more residues that make up the CH3-CH3interface in a fusion protein of the instant application are replacedwith a charged amino acid such that the interaction becomeselectrostatically unfavorable. For example, a positive-charged aminoacid in the interface (e.g., a lysine, arginine, or histidine) isreplaced with a negatively charged amino acid (e.g., aspartic acid orglutamic acid). Alternatively, or in combination with the forgoingsubstitution, a negative-charged amino acid in the interface is replacedwith a positive-charged amino acid. In certain embodiments, the aminoacid is replaced with a non-naturally occurring amino acid having thedesired charge characteristic. It should be noted that mutatingnegatively charged residues (Asp or Glu) to His will lead to increase inside chain volume, which may cause steric issues. Furthermore, Hisproton donor- and acceptor-form depends on the localized environment.These issues should be taken into consideration with the designstrategy. Because the interface residues are highly conserved in humanand mouse IgG subclasses, electrostatic steering effects disclosedherein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4.This strategy can also be extended to modifying uncharged residues tocharged residues at the CH3 domain interface.

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered to becomplementary on the basis of charge pairing (electrostatic steering).One of a pair of Fc sequences with electrostatic complementarity can bearbitrarily fused to the TGF-beta superfamily type I or type II receptorpolypeptide of the construct, with or without an optional linker, togenerate a TGF-beta superfamily type I or type II receptor fusionpolypeptide This single chain can be coexpressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multichain construct (e.g., a TGF-betasuperfamily receptor single-arm heteromeric complex). In this examplebased on electrostatic steering, SEQ ID NO: 200 [humanG1Fc(E134K/D177K)] and SEQ ID NO: 201 [human G1Fc(K170D/K187D)] areexamples of complementary Fc sequences in which the engineered aminoacid substitutions are double underlined, and the TGF-beta superfamilytype I or type II receptor polypeptide of the construct can be fused toeither SEQ ID NO: 200 or SEQ ID NO: 201, but not both. Given the highdegree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc(see FIG. 5) will generate complementary Fc pairs which may be usedinstead of the complementary hG1Fc pair below (SEQ ID NOs: 200 and 201).

(SEQ ID NO: 200) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLKSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 201) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWESNGQPENNYD TTPPVLDSDG SFFLYSDLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered forsteric complementarity. In part, the disclosure providesknobs-into-holes pairing as an example of steric complementarity. One ofa pair of Fc sequences with steric complementarity can be arbitrarilyfused to the TGF-beta superfamily type I or type II receptor polypeptideof the construct, with or without an optional linker, to generate aTGF-beta superfamily type I or type II receptor fusion polypeptide. Thissingle chain can be coexpressed in a cell of choice along with the Fcsequence complementary to the first Fc to favor generation of thedesired multichain construct (e.g., a TGF-beta superfamily receptorsingle-arm heteromeric complex). In this example based onknobs-into-holes pairing, SEQ ID NO: 202 [human G1Fc(T144Y)] and SEQ IDNO: 203 [human G1Fc(Y185T)] are examples of complementary Fc sequencesin which the engineered amino acid substitutions are double underlined,and the TGF-beta superfamily type I or type II polypeptide of theconstruct can be fused to either SEQ ID NO: 202 or SEQ ID NO: 203, butnot both. Given the high degree of amino acid sequence identity betweennative hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can beappreciated that amino acid substitutions at corresponding positions inhG2Fc, hG3Fc, or hG4Fc (see FIG. 5) will generate complementary Fc pairswhich may be used instead of the complementary hG1Fc pair below (SEQ IDNOs: 202 and 203).

(SEQ ID NO: 202) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 203) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLTSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK

An example of Fc complementarity based on knobs-into-holes pairingcombined with an engineered disulfide bond is disclosed in SEQ ID NO:204 [hG1Fc(S132C/T144W)] and SEQ ID NO: 205[hG1Fc(Y127C/T144S/L146A/Y185V)]. The engineered amino acidsubstitutions in these sequences are double underlined, and the TGF-betasuperfamily type I or type II polypeptide of the construct can be fusedto either SEQ ID NO: 204 or SEQ ID NO: 205, but not both. Given the highdegree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc(see FIG. 5) will generate complementary Fc pairs which may be usedinstead of the complementary hG1Fc pair below (SEQ ID NOs: 204 and 205).

(SEQ ID NO: 204) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 205) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered togenerate interdigitating β-strand segments of human IgG and IgA C_(H)3domains. Such methods include the use of strand-exchange engineereddomain (SEED) C_(H)3 heterodimers allowing the formation of SEEDbodyfusion proteins [see, for example, Davis et al (2010) Protein Eng DesignSel 23:195-202]. One of a pair of Fc sequences with SEEDbodycomplementarity can be arbitrarily fused to the TGF-beta superfamilytype I or type II receptor polypeptide of the construct, with or withoutan optional linker, to generate a TGF-beta superfamily type I or type IIreceptor fusion polypeptide. This single chain can be coexpressed in acell of choice along with the Fc sequence complementary to the first Fcto favor generation of the desired multichain construct. In this examplebased on SEEDbody (Sb) pairing, SEQ ID NO: 206 [hG1Fc(Sb_(AG))] and SEQID NO: 207 [hG1Fc(Sb_(GA))] are examples of complementary IgG Fcsequences in which the engineered amino acid substitutions from IgA Fcare double underlined, and the TGF-beta superfamily type I or type IIreceptor polypeptide of the construct can be fused to either SEQ ID NO:206 or SEQ ID NO: 207, but not both. Given the high degree of amino acidsequence identity between native hG1Fc, native hG2Fc, native hG3Fc, andnative hG4Fc, it can be appreciated that amino acid substitutions atcorresponding positions in hG1Fc, hG2Fc, hG3Fc, or hG4Fc (see FIG. 5)will generate an Fc monomer which may be used in the complementaryIgG-IgA pair below (SEQ ID NOs: 206 and 207).

(SEQ ID NO: 206) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PFRPEVHLLP PSREEMTKNQ VSLTCLARGF 151 YPKDIAVEWESNGQPENNYK TTPSRQEPSQ GTTTFAVTSK LTVDKSRWQQ 201 GNVFSCSVMH EALHNHYTQKTISLSPGK (SEQ ID NO: 207) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PPSEELALNE LVTLTCLVKG 151 FYPSDIAVEWESNGQELPRE KYLTWAPVLD SDGSFFLYSI LRVAAEDWKK 201 GDTFSCSVMH EALHNHYTQKSLDRSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains with a cleavable leucine zipper domainattached at the C-terminus of the Fc C_(H)3 domains. Attachment of aleucine zipper is sufficient to cause preferential assembly ofheterodimeric antibody heavy chains. See, e.g., Wranik et al (2012) JBiol Chem 287:43331-43339. As disclosed herein, one of a pair of Fcsequences attached to a leucine zipper-forming strand can be arbitrarilyfused to the TGF-beta superfamily type I or type II receptor polypeptideof the construct, with or without an optional linker, to generate aTGF-beta superfamily type I or type II receptor fusion polypeptide. Thissingle chain can be coexpressed in a cell of choice along with the Fcsequence attached to a complementary leucine zipper-forming strand tofavor generation of the desired multichain construct. Proteolyticdigestion of the construct with the bacterial endoproteinase Lys-C postpurification can release the leucine zipper domain, resulting in an Fcconstruct whose structure is identical to that of native Fc. In thisexample based on leucine zipper pairing, SEQ ID NO: 213 [hG1Fc-Apl(acidic)] and SEQ ID NO: 214 [hG1Fc-Bp1 (basic)] are examples ofcomplementary IgG Fc sequences in which the engineered complimentaryleucine zipper sequences are underlined, and the TGF-beta superfamilytype I or type II receptor polypeptide of the construct can be fused toeither SEQ ID NO: 213 or SEQ ID NO: 214, but not both. Given the highdegree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated thatleucine zipper-forming sequences attached, with or without an optionallinker, to hG1Fc, hG2Fc, hG3Fc, or hG4Fc (see FIG. 5) will generate anFc monomer which may be used in the complementary leucine zipper-formingpair below (SEQ ID NOs: 213 and 214).

(SEQ ID NO: 213) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVDVSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGKGGSAQ LEKELQALEK ENAQLEWELQ 251 ALEKELAQGA T (SEQ ID NO: 214) 1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51 VKFNWYVDGVEVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPI EKTISKAKGQPREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGKGGSAQ LKKKLQALKK KNAQLKWKLQ 251 ALKKKLAQGA T

It is understood that different elements of the fusion proteins (e.g.,immunoglobulin Fc fusion proteins) may be arranged in any manner that isconsistent with desired functionality. For example, a TGF-betasuperfamily type I or type II receptor polypeptide domain may be placedC-terminal to a heterologous domain, or alternatively, a heterologousdomain may be placed C-terminal to a TGF-beta superfamily type I or typeII receptor polypeptide domain. The TGF-beta superfamily type I or typeII receptor polypeptide domain and the heterologous domain need not beadjacent in a fusion protein, and additional domains or amino acidsequences may be included C- or N-terminal to either domain or betweenthe domains.

For example, a TGF-beta superfamily type I or type II receptor fusionpolypeptide may comprise an amino acid sequence as set forth in theformula A-B-C. The B portion corresponds to a TGF-beta superfamily typeI or type II receptor polypeptide domain. The A and C portions may beindependently zero, one, or more than one amino acid, and both the A andC portions when present are heterologous to B. The A and/or C portionsmay be attached to the B portion via a linker sequence. A linker may berich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycineand proline residues and may, for example, contain a single sequence ofthreonine/serine and glycines or repeating sequences of threonine/serineand/or glycines, e.g., GGG (SEQ ID NO: 58), GGGG (SEQ ID NO: 59), TG₄(SEQ ID NO: 60), SG₄ (SEQ ID NO: 61), TG₃ (SEQ ID NO: 62), or SG₃ (SEQID NO: 63) singlets, or repeats. In certain embodiments, a TGF-betasuperfamily type I or type II receptor fusion polypeptide comprises anamino acid sequence as set forth in the formula A-B-C, wherein A is aleader (signal) sequence, B consists of a TGF-beta superfamily type I ortype II receptor polypeptide domain, and C is a polypeptide portion thatenhances one or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, and/or purification. In certain embodiments, aTGF-beta superfamily type I or type II receptor fusion polypeptidecomprises an amino acid sequence as set forth in the formula A-B-C,wherein A is a TPA leader sequence, B consists of a TGF-beta superfamilytype I or type II receptor polypeptide domain, and C is animmunoglobulin Fc domain. Preferred fusion polypeptides comprise theamino acid sequence set forth in any one of SEQ ID NOs: 101, 103, 104,106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125,127, 128, 130, 131, 133, 134, 136, and 401-424.

In some embodiments, TGF-beta superfamily receptor single-armheteromultimer complexes of the present disclosure further comprise oneor more heterologous portions (domains) so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. Well-knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy-chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆ (SEQ ID NO: 509)) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the ligand trap polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well-known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for factor Xa orthrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation.

In certain embodiments, TGF-beta superfamily type I and/or type IIreceptor polypeptides of the present disclosure contain one or moremodifications that are capable of stabilizing the polypeptides. Forexample, such modifications enhance the in vitro half-life of thepolypeptides, enhance circulatory half-life of the polypeptides, and/orreduce proteolytic degradation of the polypeptides. Such stabilizingmodifications include, but are not limited to, fusion polypeptides(including, for example, fusion polypeptides comprising a TGF-betasuperfamily type I or type II receptor polypeptide domain and astabilizer domain), modifications of a glycosylation site (including,for example, addition of a glycosylation site to a polypeptide of thedisclosure), and modifications of carbohydrate moiety (including, forexample, removal of carbohydrate moieties from a polypeptide of thedisclosure). As used herein, the term “stabilizer domain” not onlyrefers to a fusion domain (e.g., an immunoglobulin Fc domain) as in thecase of fusion polypeptides, but also includes nonproteinaceousmodifications such as a carbohydrate moiety, or nonproteinaceous moiety,such as polyethylene glycol.

In preferred embodiments, TGF-beta superfamily receptor single-armheteromultimer complexes to be used in accordance with the methodsdescribed herein are isolated polypeptide complexes. As used herein, anisolated protein (or protein complex) or polypeptide (or polypeptidecomplex) is one which has been separated from a component of its naturalenvironment. In some embodiments, a single-arm heteromultimer complex ofthe disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99%purity as determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase HPLC). Methods forassessment of antibody purity are well known in the art [See, e.g.,Flatman et al., (2007) J. Chromatogr. B 848:79-87].

In certain embodiments, TGF-beta superfamily type I or type II receptorpolypeptides, as well as single-arm heteromultimer complexes thereof, ofthe disclosure can be produced by a variety of art-known techniques. Forexample, polypeptides of the disclosure can be synthesized usingstandard protein chemistry techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). In addition, automated peptidesynthesizers are commercially available (see, e.g., Advanced ChemTechModel 396; Milligen/Biosearch 9600). Alternatively, the polypeptides andcomplexes of the disclosure, including fragments or variants thereof,may be recombinantly produced using various expression systems [e.g., E.coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as iswell known in the art. In a further embodiment, the modified orunmodified polypeptides of the disclosure may be produced by digestionof recombinantly produced full-length TGFβ superfamily type I or type IIreceptor polypeptides by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using a commercially availablesoftware, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.)can be used to identify proteolytic cleavage sites.

3. Nucleic Acids Encoding TGFβ Superfamily Receptor Polypeptides

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding TGFβ superfamily type I or type IIreceptors (including fragments, functional variants, and fusion proteinsthereof) disclosed herein. For example, SEQ ID NO: 12 encodes thenaturally occurring human ActRIIA precursor polypeptide, while SEQ IDNO: 13 encodes the processed extracellular domain of ActRIIA. Thesubject nucleic acids may be single-stranded or double stranded. Suchnucleic acids may be DNA or RNA molecules. These nucleic acids may beused, for example, in methods for making TGF-beta superfamily single-armheteromultimer complexes of the present disclosure.

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding TGFβ superfamily type Ior type II receptor polypeptides of the present disclosure areunderstood to include nucleic acids that are variants of any one of SEQID NOs: 7, 8, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37,40, 41, 44, 45, 48, 49, 52, 53, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86,89, 90, 93, 94, 102, 105, 108, 114, 117, 120, 123, 126, 129, 132, 135,303, 304, 307, 308, 311, and 312. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions,additions, or deletions including allelic variants, and therefore, willinclude coding sequences that differ from the nucleotide sequencedesignated in any one of SEQ ID NOs: 7, 8, 12, 13, 16, 17, 20, 21, 24,25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 69, 70, 73,74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94, 102, 105, 108, 114, 117,120, 123, 126, 129, 132, 135, 303, 304, 307, 308, 311, and 312.

In certain embodiments, TGFβ superfamily type I or type II receptorpolypeptides of the present disclosure are encoded by isolated orrecombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%,97%, 98%, or 99% identical to SEQ ID NOs: 7, 8, 12, 13, 16, 17, 20, 21,24, 25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 69, 70,73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94, 102, 105, 108, 114, 117,120, 123, 126, 129, 132, 135, 303, 304, 307, 308, 311, and 312. One ofordinary skill in the art will appreciate that nucleic acid sequencesthat are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to thesequences complementary to SEQ ID NOs: 7, 8, 12, 13, 16, 17, 20, 21, 24,25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 69, 70, 73,74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94, 102, 105, 108, 114, 117,120, 123, 126, 129, 132, 135, 303, 304, 307, 308, 311, and 312 are alsowithin the scope of the present disclosure. In further embodiments, thenucleic acid sequences of the disclosure can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence or in a DNAlibrary.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under highly stringentconditions to the nucleotide sequence designated in SEQ ID NOs: 7, 8,12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45,48, 49, 52, 53, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94,102, 105, 108, 114, 117, 120, 123, 126, 129, 132, 135, 303, 304, 307,308, 311, and 312, the complement sequence of SEQ ID NOs: 7, 8, 12, 13,16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49,52, 53, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94, 102,105, 108, 114, 117, 120, 123, 126, 129, 132, 135, 303, 304, 307, 308,311, and 312, or fragments thereof. One of ordinary skill in the artwill understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 7, 8, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36,37, 40, 41, 44, 45, 48, 49, 52, 53, 69, 70, 73, 74, 77, 78, 81, 82, 85,86, 89, 90, 93, 94, 102, 105, 108, 114, 117, 120, 123, 126, 129, 132,135, 303, 304, 307, 308, 311, and 312 due to degeneracy in the geneticcode are also within the scope of the disclosure. For example, a numberof amino acids are designated by more than one triplet. Codons thatspecify the same amino acid, or synonyms (for example, CAU and CAC aresynonyms for histidine) may result in “silent” mutations which do notaffect the amino acid sequence of the protein. However, it is expectedthat DNA sequence polymorphisms that do lead to changes in the aminoacid sequences of the subject proteins will exist among mammalian cells.One skilled in the art will appreciate that these variations in one ormore nucleotides (up to about 3-5% of the nucleotides) of the nucleicacids encoding a particular protein may exist among individuals of agiven species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the presentdisclosure may be operably linked to one or more regulatory nucleotidesequences in an expression construct. Regulatory nucleotide sequenceswill generally be appropriate to the host cell used for expression.Numerous types of appropriate expression vectors and suitable regulatorysequences are known in the art for a variety of host cells. Typically,said one or more regulatory nucleotide sequences may include, but arenot limited to, promoter sequences, leader or signal sequences,ribosomal binding sites, transcriptional start and terminationsequences, translational start and termination sequences, and enhanceror activator sequences. Constitutive or inducible promoters as known inthe art are contemplated by the disclosure. The promoters may be eithernaturally occurring promoters, or hybrid promoters that combine elementsof more than one promoter. An expression construct may be present in acell on an episome, such as a plasmid, or the expression construct maybe inserted in a chromosome. In some embodiments, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the present disclosure, the subject nucleic acidis provided in an expression vector comprising a nucleotide sequenceencoding a TGFβ superfamily type I or type II receptor polypeptide andoperably linked to at least one regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe TGFβ superfamily type I or type II receptor polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, Calif. (1990). For instance, anyof a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it may be usedin these vectors to express DNA sequences encoding a TGFβ superfamilytype I or type II receptor polypeptide. Such useful expression controlsequences, include, for example, the early and late promoters of SV40,tet promoter, adenovirus or cytomegalovirus immediate early promoter,RSV promoters, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant TGFβ superfamily type I or type II receptor polypeptideinclude plasmids and other vectors. For instance, suitable vectorsinclude plasmids of the following types: pBR322-derived plasmids,pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids andpUC-derived plasmids for expression in prokaryotic cells, such as E.coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see, e.g., Molecular Cloning A LaboratoryManual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press, 2001). In some instances, it may be desirableto express the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject TGFβ superfamily type I or type II receptor polypeptide inCHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif),pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors(Promega, Madison, Wis.). As will be apparent, the subject geneconstructs can be used to cause expression of the subject TGFβsuperfamily type I or type II receptor polypeptide in cells propagatedin culture, e.g., to produce proteins, including fusion proteins orvariant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject TGFβ superfamily type I or type II receptor polypeptides. Thehost cell may be any prokaryotic or eukaryotic cell. For example, a TGFβsuperfamily type I or type II receptor polypeptide of the disclosure maybe expressed in bacterial cells such as E. coli, insect cells (e.g.,using a baculovirus expression system), yeast, or mammalian cells [e.g.a Chinese hamster ovary (CHO) cell line]. Other suitable host cells areknown to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject TGFβ superfamily type I or type II receptorpolypeptides. For example, a host cell transfected with an expressionvector encoding a TGFβ superfamily type I or type II receptorpolypeptide can be cultured under appropriate conditions to allowexpression of the TGFβ superfamily type I or type II receptorpolypeptide to occur. The polypeptide may be secreted and isolated froma mixture of cells and medium containing the polypeptide. Alternatively,the TGFβ superfamily type I or type II receptor polypeptide may beisolated from a cytoplasmic or membrane fraction obtained from harvestedand lysed cells. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The subject polypeptides can be isolated from cell culture medium, hostcells, or both, using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, immunoaffinitypurification with antibodies specific for particular epitopes of theTGFβ superfamily type I or type II receptor polypeptides and affinitypurification with an agent that binds to a domain fused to TGFβsuperfamily type I or type II receptor polypeptide (e.g., a protein Acolumn may be used to purify a TGFβ superfamily type I receptor-Fc ortype II receptor-Fc fusion polypeptide or protein complex). In someembodiments, the TGFβ superfamily type I or type II receptor polypeptideis a fusion polypeptide or protein complex containing a domain whichfacilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. A TGFβsuperfamily type I receptor-Fc or type II receptor-Fc fusion polypeptideor protein complex may be purified to a purityof >90%, >95%, >96%, >98%, or >99% as determined by size exclusionchromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDSPAGE. The target level of purity should be one that is sufficient toachieve desirable results in mammalian systems, particularly non-humanprimates, rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant TGFβsuperfamily type I or type II receptor polypeptide, can allowpurification of the expressed fusion protein by affinity chromatographyusing a Ni²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase to provide thepurified TGFβ superfamily type I or type II receptor polypeptide orprotein complex. See, e.g., Hochuli et al. (1987) J Chromatography411:177; and Janknecht et al. (1991) PNAS USA 88:8972.

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

4. Screening Assays

In certain aspects, the present disclosure relates to the use of TGFβsuperfamily type I and type II receptor single-arm heteromultimercomplexes to identify compounds (agents) which are agonists orantagonists of TGFβ superfamily receptors. Compounds identified throughthis screening can be tested to assess their ability to modulate tissuessuch as bone, cartilage, muscle, fat, and/or neurons, to assess theirability to modulate tissue growth in vivo or in vitro. These compoundscan be tested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting TGFβ superfamily ligand signaling(e.g., SMAD 2/3 and/or SMAD 1/5/8 signaling). In certain embodiments,high-throughput screening of compounds can be carried out to identifyagents that perturb TGFβ superfamily receptor-mediated effects on aselected cell line. In certain embodiments, the assay is carried out toscreen and identify compounds that specifically inhibit or reducebinding of a TGF-beta superfamily receptor single-arm heteromultimercomplex to its binding partner, such as a TGFβ superfamily ligand (e.g.,BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9,BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11,GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C,activin E, activin AB, activin AC, activin AE, activin BC, activin BE,nodal, GDNF, neurturin, artemin, persephin, MIS, and Lefty).Alternatively, the assay can be used to identify compounds that enhancebinding of a TGF-beta superfamily receptor single-arm heteromultimercomplex to its binding partner such as an TGFβ superfamily ligand. In afurther embodiment, the compounds can be identified by their ability tointeract with a TGF-beta superfamily receptor single-arm heteromultimercomplex of the disclosure.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Incertain embodiments, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the disclosure can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S-transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug-screening programs which test libraries of compounds andnatural extracts, high-throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a TGF-betasuperfamily receptor single-arm heteromultimer complex and its bindingpartner (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7,BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8,GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A,activin B, activin C, activin E, activin AB, activin AC, activin AE,activin BC, activin BE, nodal, GDNF, neurturin, artemin, persephin, MIS,and Lefty).

Merely to illustrate, in an exemplary screening assay of the presentdisclosure, the compound of interest is contacted with an isolated andpurified TGF-beta superfamily receptor single-arm heteromultimer complexwhich is ordinarily capable of binding to a TGF-beta superfamily ligand,as appropriate for the intention of the assay. To the mixture of thecompound and TGF-beta superfamily receptor single-arm heteromultimercomplex is then added the appropriate TGF-beta superfamily ligand (e.g.,BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9,BMP10, GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11,GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C,activin E, activin AB, activin AC, activin AE, activin BC, activin BE,nodal, GDNF, neurturin, artemin, persephin, MIS, and Lefty). Detectionand quantification of complexes between single-arm heteromultimers andsuperfamily ligands provides a means for determining the compound'sefficacy at inhibiting (or potentiating) complex formation between theTGF-beta superfamily receptor single-arm heteromultimer complex and itsbinding protein. The efficacy of the compound can be assessed bygenerating dose-response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. For example, in acontrol assay, isolated and purified TGF-beta superfamily ligand isadded to a composition containing the TGF-beta superfamily receptorsingle-arm heteromultimer complex, and the formation ofheteromultimer-ligand complex is quantitated in the absence of the testcompound. It will be understood that, in general, the order in which thereactants may be admixed can be varied, and can be admixedsimultaneously. Moreover, in place of purified proteins, cellularextracts and lysates may be used to render a suitable cell-free assaysystem.

Binding of a TGF-beta superfamily receptor single-arm heteromultimercomplex to another protein may be detected by a variety of techniques.For instance, modulation of the formation of complexes can bequantitated using, for example, detectably labeled proteins such asradiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g.,FITC), or enzymatically labeled TGF-beta superfamily receptor single-armheteromultimer complex and its binding protein by immunoassay or bychromatographic detection.

In certain embodiments, the present disclosure contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a TGF-beta superfamily receptor single-armheteromultimer complex and its binding protein. Further, other modes ofdetection, such as those based on optical waveguides (see, e.g., PCTPublication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmonresonance (SPR), surface charge sensors, and surface force sensors, arecompatible with many embodiments of the disclosure.

Moreover, the present disclosure contemplates the use of an interactiontrap assay, also known as the “two-hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a TGF-beta superfamilyreceptor single-arm heteromultimer complex and its binding partner. See,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). In a specific embodiment, the present disclosurecontemplates the use of reverse two-hybrid systems to identify compounds(e.g., small molecules or peptides) that dissociate interactions betweena TGF-beta superfamily receptor single-arm heteromultimer complex andits binding protein [see, e.g., Vidal and Legrain, (1999) Nucleic AcidsRes 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81;and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368].

In certain embodiments, the subject compounds are identified by theirability to interact with a TGF-beta superfamily receptor single-armheteromultimer complex of the disclosure. The interaction between thecompound and the TGF-beta superfamily receptor single-arm heteromultimercomplex may be covalent or non-covalent. For example, such interactioncan be identified at the protein level using in vitro biochemicalmethods, including photo-crosslinking, radiolabeled ligand binding, andaffinity chromatography. See, e.g., Jakoby W B et al. (1974) Methods inEnzymology 46:1. In certain cases, the compounds may be screened in amechanism-based assay, such as an assay to detect compounds which bindto a TGF-beta superfamily receptor single-arm heteromultimer complex.This may include a solid-phase or fluid-phase binding event.Alternatively, the gene encoding a TGF-beta superfamily receptorsingle-arm heteromultimer complex can be transfected with a reportersystem (e.g., β-galactosidase, luciferase, or green fluorescent protein)into a cell and screened against the library preferably byhigh-throughput screening or with individual members of the library.Other mechanism-based binding assays may be used; for example, bindingassays which detect changes in free energy. Binding assays can beperformed with the target fixed to a well, bead or chip or captured byan immobilized antibody or resolved by capillary electrophoresis. Thebound compounds may be detected usually using colorimetric endpoints orfluorescence or surface plasmon resonance.

5. Exemplary Therapeutic Uses

In certain embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the present disclosure can beused to treat or prevent a disease or condition that is associated withabnormal activity of a TGFβ superfamily receptor (e.g., ALK1, ALK2,ALK3, ALK4, ALK5, ALK6, ALK7, ActRIIA, ActRIIB, BMPRII, TGFBRII, andMISRII) and/or a TGFβ superfamily ligand (e.g., BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, activin AE, activin BC, activin BE, nodal, GDNF, neurturin,artemin, persephin, MIS, and Lefty). These diseases, disorders orconditions are generally referred to herein as “TGFβsuperfamily-associated conditions.” In certain embodiments, the presentinvention provides methods of treating or preventing an individual inneed thereof through administering to the individual a therapeuticallyeffective amount of a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, as described herein. The terms“subject,” an “individual,” or a “patient” are interchangeablethroughout the specification. Any of the TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the present disclosure canpotentially be employed individually or in combination for therapeuticuses disclosed herein. These methods are particularly aimed attherapeutic and prophylactic treatments of mammals including, forexample, rodents, primates, and humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician or other health care provider andthe intended result of administration of the therapeutic agent.

Native TGFβ superfamily receptor-ligand complexes play essential rolesin tissue growth as well as early developmental processes such as thecorrect formation of various structures or in one or morepost-developmental capacities including sexual development, pituitaryhormone production, and creation of bone and cartilage. Thus, TGFβsuperfamily-associated conditions/disorders include abnormal tissuegrowth and developmental defects. In addition, TGFβsuperfamily-associated conditions include, but are not limited to,disorders of cell growth and differentiation such as inflammation,allergy, autoimmune diseases, infectious diseases, and tumors.

Exemplary TGFβ superfamily-associated conditions include neuromusculardisorders (e.g., muscular dystrophy and muscle atrophy), congestiveobstructive pulmonary disease (and muscle wasting associated with COPD),muscle wasting syndrome, sarcopenia, cachexia, adipose tissue disorders(e.g., obesity), type 2 diabetes (NIDDM, adult-onset diabetes), and bonedegenerative disease (e.g., osteoporosis). Other exemplary TGFβsuperfamily-associated conditions include musculodegenerative andneuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis), andimmunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes).

In certain embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the disclosure are used as partof a treatment for a muscular dystrophy. The term “muscular dystrophy”refers to a group of degenerative muscle diseases characterized bygradual weakening and deterioration of skeletal muscles and sometimesthe heart and respiratory muscles. Muscular dystrophies are geneticdisorders characterized by progressive muscle wasting and weakness thatbegin with microscopic changes in the muscle. As muscles degenerate overtime, the person's muscle strength declines. Exemplary musculardystrophies that can be treated with a regimen including the subjectTGF-beta superfamily receptor single-arm heteromultimer complexesinclude: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy(BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle musculardystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD)(also known as Landouzy-Dejerine), myotonic dystrophy (MMD; also knownas Steinert's Disease), oculopharyngeal muscular dystrophy (OPMD),distal muscular dystrophy (DD), congenital muscular dystrophy (CMD).

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. Beckermuscular dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is defective. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either of insufficient quantityor poor quality. The presence of some dystrophin protects the muscles ofpatients with BMD from degenerating as severely or as quickly as thoseof patients with DMD.

Studies in animals indicate that inhibition of the GDF8 signalingpathway may effectively treat various aspects of disease in DMD and BMDpatients (Bogdanovich et al., 2002, Nature 420:418-421; Pistilli et al.,2011, Am J Pathol 178:1287-1297). Thus, TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may act as GDF8inhibitors (antagonists), and constitute an alternative means ofblocking signaling by GDF8 and/or related TGFβ superfamily ligands invivo in DMD and BMD patients.

Similarly, TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure may provide an effective means to increasemuscle mass in other disease conditions that are in need of musclegrowth. For example, amyotrophic lateral sclerosis (ALS), also calledLou Gehrig's disease or motor neuron disease, is a chronic, progressive,and incurable CNS disorder that attacks motor neurons, which arecomponents of the central nervous system required for initiation ofskeletal muscle contraction. In ALS, motor neurons deteriorate andeventually die, and though a person's brain normally remains fullyfunctioning and alert, initiation of muscle contraction is blocked atthe spinal level. Individuals who develop ALS are typically between 40and 70 years old, and the first motor neurons to degenerate are thoseinnervating the arms or legs. Patients with ALS may have troublewalking, may drop things, fall, slur their speech, and laugh or cryuncontrollably. As the disease progresses, muscles in the limbs begin toatrophy from disuse. Muscle weakness becomes debilitating, and patientseventually require a wheel chair or become confined to bed. Most ALSpatients die from respiratory failure or from complications ofventilator assistance like pneumonia 3-5 years from disease onset.

Promotion of increased muscle mass by TGF-beta superfamily receptorsingle-arm heteromultimer complexes might also benefit those sufferingfrom muscle wasting diseases. Gonzalez-Cadavid et al. (supra) reportedthat GDF8 expression correlates inversely with fat-free mass in humansand that increased expression of the GDF8 gene is associated with weightloss in men with AIDS wasting syndrome. By inhibiting the function ofGDF8 in AIDS patients, at least certain symptoms of AIDS may bealleviated, if not completely eliminated, thus significantly improvingquality of life in AIDS patients.

Since loss of GDF8 function is also associated with fat loss withoutdiminution of nutrient intake (Zimmers et al., supra; McPherron and Lee,supra), the subject TGF-beta superfamily receptor single-armheteromultimer complexes may further be used as a therapeutic agent forslowing or preventing the development of obesity and type 2 diabetes.

Cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. This syndrome is a common feature ofmany types of cancer—present in approximately 80% of cancer patients atdeath—and is responsible not only for a poor quality of life and poorresponse to chemotherapy but also a shorter survival time than is foundin patients with comparable tumors but without weight loss. Cachexia istypically suspected in patients with cancer if an involuntary weightloss of greater than five percent of premorbid weight occurs within asix-month period. Associated with anorexia, wasting of fat and muscletissue, and psychological distress, cachexia arises from a complexinteraction between the cancer and the host. Cancer cachexia affectscytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Currently, there is no treatment to control or reversethe cachexic process. Since systemic overexpression of GDF8 in adultmice was found to induce profound muscle and fat loss analogous to thatseen in human cachexia syndromes (Zimmers et al., supra), the subjectTGF-beta superfamily receptor single-arm heteromultimer complexpharmaceutical compositions may be beneficially used to prevent, treat,or alleviate the symptoms of the cachexia syndrome, where muscle growthis desired.

In certain embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the present disclosure may beused in methods of inducing bone and/or cartilage formation, preventingbone loss, increasing bone mineralization, preventing thedemineralization of bone, and/or increasing bone density. TGF-betasuperfamily receptor single-arm heteromultimer complexes may be usefulin patients who are diagnosed with subclinical low bone density, as aprotective measure against the development of osteoporosis.

In some embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the present disclosure may findmedical utility in the healing of bone fractures and cartilage defectsin humans and other animals. The subject methods and compositions mayalso have prophylactic use in closed as well as open fracture reductionand also in the improved fixation of artificial joints. De novo boneformation induced by an osteogenic agent is useful for repair ofcraniofacial defects that are congenital, trauma-induced, or caused byoncologic resection, and is also useful in cosmetic plastic surgery.Further, methods and compositions of the invention may be used in thetreatment of periodontal disease and in other tooth repair processes. Incertain cases, a TGF-beta superfamily receptor single-arm heteromultimercomplex, or combinations of TGF-beta superfamily receptor single-armheteromultimer complexes, may provide an environment to attractbone-forming cells, stimulate growth of bone-forming cells, or inducedifferentiation of progenitors of bone-forming cells. TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure may also be useful in the treatment of osteoporosis. Further,TGF-beta superfamily receptor single-arm heteromultimer complexes may beused in repair of cartilage defects and prevention/reversal ofosteoarthritis.

Rosen et al. (ed) Primer on the Metabolic Bone Diseases and Disorders ofMineral Metabolism, 7^(th) ed. American Society for Bone and MineralResearch, Washington D.C. (incorporated herein by reference) provides anextensive discussion of bone disorders that may be subject to treatmentwith a TGF-beta superfamily receptor single-arm heteromultimer complexor with combinations of TGF-beta superfamily receptor single-armheteromultimer complexes. A partial listing is provided herein. Methodsand compositions of the invention can be applied to conditionscharacterized by or causing bone loss, such as osteoporosis (includingsecondary osteoporosis), hyperparathyroidism, chronic kidney diseasemineral bone disorder, sex hormone deprivation or ablation (e.g.androgen and/or estrogen), glucocorticoid treatment, rheumatoidarthritis, severe burns, hyperparathyroidism, hypercalcemia,hypocalcemia, hypophosphatemia, osteomalacia (including tumor-inducedosteomalacia), hyperphosphatemia, vitamin D deficiency,hyperparathyroidism (including familial hyperparathyroidism) andpseudohypoparathyroidism, tumor metastases to bone, bone loss as aconsequence of a tumor or chemotherapy, tumors of the bone and bonemarrow (e.g., multiple myeloma), ischemic bone disorders, periodontaldisease and oral bone loss, Cushing's disease, Paget's disease,thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubularacidosis, or anorexia nervosa. Methods and compositions of the inventionmay also be applied to conditions characterized by a failure of boneformation or healing, including non-union fractures, fractures that areotherwise slow to heal, fetal and neonatal bone dysplasias (e.g.,hypocalcemia, hypercalcemia, calcium receptor defects and vitamin Ddeficiency), osteonecrosis (including osteonecrosis of the jaw) andosteogenesis imperfecta. Additionally, the anabolic effects will causesuch antagonists to diminish bone pain associated with bone damage orerosion. As a consequence of the anti-resorptive effects, suchantagonists may be useful to treat disorders of abnormal bone formation,such as osteoblastic tumor metastases (e.g., associated with primaryprostate or breast cancer), osteogenic osteosarcoma, osteopetrosis,progressive diaphyseal dysplasia, endosteal hyperostosis,osteopoikilosis, and melorheostosis. Other disorders that may be treatedinclude fibrous dysplasia and chondrodysplasias.

In another specific embodiment, the disclosure provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See, e.g., PCT PublicationNo. WO 84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure in admixture with apharmaceutically acceptable vehicle, carrier, or matrix.

In some embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the disclosure can be applied toconditions causing bone loss such as osteoporosis, hyperparathyroidism,Cushing's disease, thyrotoxicosis, chronic diarrheal state ormalabsorption, renal tubular acidosis, or anorexia nervosa. It iscommonly appreciated that being female, having a low body weight, andleading a sedentary lifestyle are risk factors for osteoporosis (loss ofbone mineral density, leading to fracture risk). However, osteoporosiscan also result from the long-term use of certain medications.Osteoporosis resulting from drugs or another medical condition is knownas secondary osteoporosis. In Cushing's disease, the excess amount ofcortisol produced by the body results in osteoporosis and fractures. Themost common medications associated with secondary osteoporosis are thecorticosteroids, a class of drugs that act like cortisol, a hormoneproduced naturally by the adrenal glands. Although adequate levels ofthyroid hormones are needed for the development of the skeleton, excessthyroid hormone can decrease bone mass over time. Antacids that containaluminum can lead to bone loss when taken in high doses by people withkidney problems, particularly those undergoing dialysis. Othermedications that can cause secondary osteoporosis include phenytoin(Dilantin) and barbiturates that are used to prevent seizures;methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for some forms ofarthritis, cancer, and immune disorders; cyclosporine (Sandimmune,Neoral), a drug used to treat some autoimmune diseases and to suppressthe immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Bone loss resulting fromcancer therapy is widely recognized and termed cancer therapy-inducedbone loss (CTIBL). Bone metastases can create cavities in the bone thatmay be corrected by treatment with a TGF-beta superfamily heteromultimercomplex. Bone loss can also be caused by gum disease, a chronicinfection in which bacteria located in gum recesses produce toxins andharmful enzymes.

In a further embodiment, the present disclosure provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients with thecongenital disorder fibrodysplasia ossificans progressiva (FOP) areafflicted by progressive ectopic bone growth in soft tissuesspontaneously or in response to tissue trauma, with a major impact onquality of life. Additionally, abnormal bone growth can occur after hipreplacement surgery and thus ruin the surgical outcome. This is a morecommon example of pathological bone growth and a situation in which thesubject methods and compositions may be therapeutically useful. The samemethods and compositions may also be useful for treating other forms ofabnormal bone growth (e.g., pathological growth of bone followingtrauma, burns or spinal cord injury), and for treating or preventing theundesirable conditions associated with the abnormal bone growth seen inconnection with metastatic prostate cancer or osteosarcoma.

In certain embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the disclosure may be used topromote bone formation in patients with cancer. Patients having certaintumors (e.g. prostate, breast, multiple myeloma or any tumor causinghyperparathyroidism) are at high risk for bone loss due to tumor-inducedbone loss, bone metastases, and therapeutic agents. Such patients may betreated with a TGF-beta superfamily receptor single-arm heteromultimercomplex, or a combination of complexes, even in the absence of evidenceof bone loss or bone metastases. Patients may also be monitored forevidence of bone loss or bone metastases, and may be treated with aTGF-beta superfamily receptor single-arm heteromultimer complex in theevent that indicators suggest an increased risk. Generally, DEXA scansare employed to assess changes in bone density, while indicators of boneremodeling may be used to assess the likelihood of bone metastases.Serum markers may be monitored. Bone specific alkaline phosphatase(BSAP) is an enzyme that is present in osteoblasts. Blood levels of BSAPare increased in patients with bone metastasis and other conditions thatresult in increased bone remodeling. Osteocalcin and procollagenpeptides are also associated with bone formation and bone metastases.Increases in BSAP have been detected in patients with bone metastasiscaused by prostate cancer, and to a lesser degree, in bone metastasesfrom breast cancer. BMP7 levels are high in prostate cancer that hasmetastasized to bone, but not in bone metastases due to bladder, skin,liver, or lung cancer. Type I carboxy-terminal telopeptide (ICTP) is acrosslink found in collagen that is formed during to the resorption ofbone. Since bone is constantly being broken down and reformed, ICTP willbe found throughout the body. However, at the site of bone metastasis,the level will be significantly higher than in an area of normal bone.ICTP has been found in high levels in bone metastasis due to prostate,lung, and breast cancer. Another collagen crosslink, Type I N-terminaltelopeptide (NTx), is produced along with ICTP during bone turnover. Theamount of NTx is increased in bone metastasis caused by many differenttypes of cancer including lung, prostate, and breast cancer. Also, thelevels of NTx increase with the progression of the bone metastasis.Therefore, this marker can be used to both detect metastasis as well asmeasure the extent of the disease. Other markers of resorption includepyridinoline and deoxypyridinoline. Any increase in resorption markersor markers of bone metastases indicate the need for therapy with aTGF-beta superfamily receptor single-arm heteromultimer complex, orcombinations of TGF-beta superfamily receptor single-arm heteromultimercomplexes, in a patient.

In another embodiment, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, may be used in patients withchronic kidney disease mineral bone disorder (CKD-MBD), a broad syndromeof interrelated skeletal, cardiovascular, and mineral-metabolicdisorders arising from kidney disease. CKD-MBD encompasses variousskeletal pathologies often referred to as renal osteodystrophy (ROD),which is a preferred embodiment for treatment with a TGF-betasuperfamily receptor single-arm heteromultimer complex, or combinationsof TGF-beta superfamily receptor single-arm heteromultimer complexes.Depending on the relative contribution of different pathogenic factors,ROD is manifested as diverse pathologic patterns of bone remodeling(Hruska et al., 2008, Chronic kidney disease mineral bone disorder(CKD-MBD); in Rosen et al. (ed) Primer on the Metabolic Bone Diseasesand Disorders of Mineral Metabolism, 7th ed. American Society for Boneand Mineral Research, Washington D.C., pp 343-349). At one end of thespectrum is ROD with uremic osteodystrophy and low bone turnover,characterized by a low number of active remodeling sites, profoundlysuppressed bone formation, and low bone resorption. At the other extremeis ROD with hyperparathyroidism, high bone turnover, and osteitisfibrosa. Given that a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, may exert both anabolic andantiresorptive effects, these agents may be useful in patients acrossthe ROD pathology spectrum.

A TGF-beta superfamily receptor single-arm heteromultimer complex, orcombinations of TGF-beta superfamily receptor single-arm heteromultimercomplexes, of the disclosure may be conjointly administered with otherbone-active pharmaceutical agents. Conjoint administration may beaccomplished by administration of a single co-formulation, bysimultaneous administration, or by administration at separate times.TGF-beta superfamily receptor single-arm heteromultimer complexes may beparticularly advantageous if administered with other bone-active agents.A patient may benefit from conjointly receiving a TGF-beta superfamilyreceptor single-arm heteromultimer complex and taking calciumsupplements, vitamin D, appropriate exercise and/or, in some cases,other medication. Examples of other medications include, bisphosphonates(alendronate, ibandronate and risedronate), calcitonin, estrogens,parathyroid hormone and raloxifene. The bisphosphonates (alendronate,ibandronate and risedronate), calcitonin, estrogens and raloxifeneaffect the bone remodeling cycle and are classified as anti-resorptivemedications. Bone remodeling consists of two distinct stages: boneresorption and bone formation. Anti-resorptive medications slow or stopthe bone-resorbing portion of the bone-remodeling cycle but do not slowthe bone-forming portion of the cycle. As a result, new formationcontinues at a greater rate than bone resorption, and bone density mayincrease over time. Teriparatide, a form of parathyroid hormone,increases the rate of bone formation in the bone remodeling cycle.Alendronate is approved for both the prevention (5 mg per day or 35 mgonce a week) and treatment (10 mg per day or 70 mg once a week) ofpostmenopausal osteoporosis. Alendronate reduces bone loss, increasesbone density and reduces the risk of spine, wrist and hip fractures.Alendronate also is approved for treatment of glucocorticoid-inducedosteoporosis in men and women as a result of long-term use of thesemedications (i.e., prednisone and cortisone) and for the treatment ofosteoporosis in men. Alendronate plus vitamin D is approved for thetreatment of osteoporosis in postmenopausal women (70 mg once a weekplus vitamin D), and for treatment to improve bone mass in men withosteoporosis. Ibandronate is approved for the prevention and treatmentof postmenopausal osteoporosis. Taken as a once-a-month pill (150 mg),ibandronate should be taken on the same day each month. Ibandronatereduces bone loss, increases bone density and reduces the risk of spinefractures. Risedronate is approved for the prevention and treatment ofpostmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mgdose or 35 mg dose with calcium), risedronate slows bone loss, increasesbone density and reduces the risk of spine and non-spine fractures.Risedronate also is approved for use by men and women to prevent and/ortreat glucocorticoid-induced osteoporosis that results from long-termuse of these medications (i.e., prednisone or cortisone). Calcitonin isa naturally occurring hormone involved in calcium regulation and bonemetabolism. In women who are more than 5 years beyond menopause,calcitonin slows bone loss, increases spinal bone density, and mayrelieve the pain associated with bone fractures. Calcitonin reduces therisk of spinal fractures. Calcitonin is available as an injection(50-100 IU daily) or nasal spray (200 IU daily).

A patient may also benefit from conjointly receiving a TGF-betasuperfamily receptor single-arm heteromultimer complex, or combinationsof TGF-beta superfamily receptor single-arm heteromultimer complexes,and additional bone-active medications. Estrogen therapy (ET)/hormonetherapy (HT) is approved for the prevention of osteoporosis. ET has beenshown to reduce bone loss, increase bone density in both the spine andhip, and reduce the risk of hip and spinal fractures in postmenopausalwomen. ET is administered most commonly in the form of a pill or skinpatch that delivers a low dose of approximately 0.3 mg daily or astandard dose of approximately 0.625 mg daily and is effective even whenstarted after age 70. When estrogen is taken alone, it can increase awoman's risk of developing cancer of the uterine lining (endometrialcancer). To eliminate this risk, healthcare providers prescribe thehormone progestin in combination with estrogen (hormone replacementtherapy or HT) for those women who have an intact uterus. ET/HT relievesmenopause symptoms and has been shown to have a beneficial effect onbone health. Side effects may include vaginal bleeding, breasttenderness, mood disturbances and gallbladder disease. Raloxifene, 60 mga day, is approved for the prevention and treatment of postmenopausalosteoporosis. It is from a class of drugs called selective estrogenreceptor modulators (SERMs) that have been developed to provide thebeneficial effects of estrogens without their potential disadvantages.Raloxifene increases bone mass and reduces the risk of spine fractures.Data are not yet available to demonstrate that raloxifene can reduce therisk of hip and other non-spine fractures. Teriparatide, a form ofparathyroid hormone, is approved for the treatment of osteoporosis inpostmenopausal women and men who are at high risk for a fracture. Thismedication stimulates new bone formation and significantly increasesbone mineral density. In postmenopausal women, fracture reduction wasnoted in the spine, hip, foot, ribs and wrist. In men, fracturereduction was noted in the spine, but there were insufficient data toevaluate fracture reduction at other sites. Teriparatide isself-administered as a daily injection for up to 24 months.

In other embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes can be used for regulating body fatcontent in an animal and for treating or preventing conditions relatedthereto, and particularly, health-compromising conditions relatedthereto. According to the present invention, to regulate (control) bodyweight can refer to reducing or increasing body weight, reducing orincreasing the rate of weight gain, or increasing or reducing the rateof weight loss, and also includes actively maintaining, or notsignificantly changing body weight (e.g., against external or internalinfluences which may otherwise increase or decrease body weight). Oneembodiment of the present disclosure relates to regulating body weightby administering to an animal (e.g., a human) in need thereof a TGF-betasuperfamily receptor single-arm heteromultimer complex, or combinationsof TGF-beta superfamily receptor single-arm heteromultimer complexese,of the disclosure.

In some embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or combinations of TGF-beta superfamily receptorsingle-arm heteromultimer complexes, of the present disclosure can beused for reducing body weight and/or reducing weight gain in an animal,and more particularly, for treating or ameliorating obesity in patientsat risk for or suffering from obesity. In another specific embodiment,the present invention is directed to methods and compounds for treatingan animal that is unable to gain or retain weight (e.g., an animal witha wasting syndrome). Such methods are effective to increase body weightand/or mass, or to reduce weight and/or mass loss, or to improveconditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass. In addition, disorders of highcholesterol (e.g., hypercholesterolemia or dislipidemia) may be treatedwith a TGF-beta superfamily receptor single-arm heteromultimer complex,or combinations of TGF-beta superfamily receptor single-armheteromultimer complexes, of the disclosure.

In certain aspects, a TGF-beta superfamily receptor single-armheteromultimer complex, or a combination of TGF-beta superfamilyreceptor single-arm heteromultimer complexes, of the present disclosurecan be used to increase red blood cell levels, treat or prevent ananemia, and/or treat or prevent ineffective erythropoiesis in a subjectin need thereof. In certain aspects, a TGF-beta superfamily receptorsingle-arm heteromultimer complex, or a combination of TGF-betasuperfamily receptor single-arm heteromultimer complexes, of the presentdisclosure may be used in combination with conventional therapeuticapproaches for increasing red blood cell levels, particularly those usedto treat anemias of multifactorial origin. Conventional therapeuticapproaches for increasing red blood cell levels include, for example,red blood cell transfusion, administration of one or more EPO receptoractivators, hematopoietic stem cell transplantation, immunosuppressivebiologics and drugs (e.g., corticosteroids). In certain embodiments, aTGF-beta superfamily receptor single-arm heteromultimer complex, or acombination of TGF-beta superfamily receptor single-arm heteromultimercomplexes, of the present disclosure can be used to treat or preventineffective erythropoiesis and/or the disorders associated withineffective erythropoiesis in a subject in need thereof. In certainaspects, a TGF-beta superfamily receptor single-arm heteromultimercomplex, or a combination of TGF-beta superfamily receptor single-armheteromultimer complexes, of the present disclosure can be used incombination with conventional therapeutic approaches for treating orpreventing an anemia or ineffective erythropoiesis disorder,particularly those used to treat anemias of multifactorial origin.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure is achieved by administering aTGF-beta superfamily receptor single-arm heteromultimer complex, or acombination of TGF-beta superfamily receptor single-arm heteromultimercomplexes, of the present disclosure in an “effective amount”. Aneffective amount of an agent refers to an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. A “therapeutically effective amount” of an agent ofthe present disclosure may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theagent to elicit a desired response in the individual. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result.

In certain embodiments, a TGF-beta superfamily receptor single-armheteromultimer complex, or a combination of TGF-beta superfamilyreceptor single-arm heteromultimer complexes, optionally combined withan EPO receptor activator, may be used to increase red blood cell,hemoglobin, or reticulocyte levels in healthy individuals and selectedpatient populations. Examples of appropriate patient populations includethose with undesirably low red blood cell or hemoglobin levels, such aspatients having an anemia, and those that are at risk for developingundesirably low red blood cell or hemoglobin levels, such as thosepatients who are about to undergo major surgery or other procedures thatmay result in substantial blood loss. In one embodiment, a patient withadequate red blood cell levels is treated with a TGF-beta superfamilyreceptor single-arm heteromultimer complex, or a combination of TGF-betasuperfamily receptor single-arm heteromultimer complexes, to increasered blood cell levels, and then blood is drawn and stored for later usein transfusions.

One or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure, optionally combined with an EPO receptoractivator, may be used to increase red blood cell levels, hemoglobinlevels, and/or hematocrit levels in a patient having an anemia. Whenobserving hemoglobin and/or hematocrit levels in humans, a level of lessthan normal for the appropriate age and gender category may beindicative of anemia, although individual variations are taken intoaccount. For example, a hemoglobin level from 10-12.5 g/dl, andtypically about 11.0 g/dl is considered to be within the normal range inhealth adults, although, in terms of therapy, a lower target level maycause fewer cardiovascular side effects [see, e.g., Jacobs et al. (2000)Nephrol Dial Transplant 15, 15-19]. Alternatively, hematocrit levels(percentage of the volume of a blood sample occupied by the cells) canbe used as a measure for anemia. Hematocrit levels for healthyindividuals range from about 41-51% for adult males and from 35-45% foradult females. In certain embodiments, a patient may be treated with adosing regimen intended to restore the patient to a target level of redblood cells, hemoglobin, and/or hematocrit. As hemoglobin and hematocritlevels vary from person to person, optimally, the target hemoglobinand/or hematocrit level can be individualized for each patient.

Anemia is frequently observed in patients having a tissue injury, aninfection, and/or a chronic disease, particularly cancer. In somesubjects, anemia is distinguished by low erythropoietin levels and/or aninadequate response to erythropoietin in the bone marrow [see, e.g.,Adamson (2008) Harrison's Principles of Internal Medicine, 17th ed.;McGraw Hill, New York, pp 628-634]. Potential causes of anemia include,for example, blood loss, nutritional deficits (e.g. reduced dietaryintake of protein), medication reaction, various problems associatedwith the bone marrow, and many diseases. More particularly, anemia hasbeen associated with a variety of disorders and conditions that include,for example, bone marrow transplantation; solid tumors (e.g., breastcancer, lung cancer, and colon cancer); tumors of the lymphatic system(e.g., chronic lymphocyte leukemia, non-Hodgkins lymphoma, and Hodgkinslymphoma); tumors of the hematopoietic system (e.g., leukemia, amyelodysplastic syndrome and multiple myeloma); radiation therapy;chemotherapy (e.g., platinum containing regimens); inflammatory andautoimmune diseases, including, but not limited to, rheumatoidarthritis, other inflammatory arthritides, systemic lupus erythematosis(SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatorybowel disease (e.g., Crohn's disease and ulcerative colitis); acute orchronic renal disease or failure, including idiopathic or congenitalconditions; acute or chronic liver disease; acute or chronic bleeding;situations where transfusion of red blood cells is not possible due topatient allo- or auto-antibodies and/or for religious reasons (e.g.,some Jehovah's Witnesses); infections (e.g., malaria and osteomyelitis);hemoglobinopathies including, for example, sickle cell disease (anemia),thalassemias; drug use or abuse (e.g., alcohol misuse); pediatricpatients with anemia from any cause to avoid transfusion; and elderlypatients or patients with underlying cardiopulmonary disease with anemiawho cannot receive transfusions due to concerns about circulatoryoverload [see, e.g., Adamson (2008) Harrison's Principles of InternalMedicine, 17th ed.; McGraw Hill, New York, pp 628-634].

Many factors can contribute to cancer-related anemia. Some areassociated with the disease process itself and the generation ofinflammatory cytokines such as interleukin-1, interferon-gamma, andtumor necrosis factor [Bron et al. (2001) Semin Oncol 28(Suppl 8):1-6].Among its effects, inflammation induces the key iron-regulatory peptidehepcidin, thereby inhibiting iron export from macrophages and generallylimiting iron availability for erythropoiesis [see, e.g., Ganz (2007) JAm Soc Nephrol 18:394-400]. Blood loss through various routes can alsocontribute to cancer-related anemia. The prevalence of anemia due tocancer progression varies with cancer type, ranging from 5% in prostatecancer up to 90% in multiple myeloma. Cancer-related anemia has profoundconsequences for patients, including fatigue and reduced quality oflife, reduced treatment efficacy, and increased mortality. In someembodiments, one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure, optionally combined with anEPO receptor activator, could be used to treat a cancer-related anemia.

A hypoproliferative anemia can result from primary dysfunction orfailure of the bone marrow. Hypoproliferative anemias include: anemia ofchronic disease, anemia of kidney disease, anemia associated withhypometabolic states, and anemia associated with cancer. In each ofthese types, endogenous erythropoietin levels are inappropriately lowfor the degree of anemia observed. Other hypoproliferative anemiasinclude: early-stage iron-deficient anemia, and anemia caused by damageto the bone marrow. In these types, endogenous erythropoietin levels areappropriately elevated for the degree of anemia observed. Prominentexamples would be myelosuppression caused by cancer and/orchemotherapeutic drugs or cancer radiation therapy. A broad review ofclinical trials found that mild anemia can occur in 100% of patientsafter chemotherapy, while more severe anemia can occur in up to 80% ofsuch patients [see, e.g., Groopman et al. (1999) J Natl Cancer Inst91:1616-1634]. Myelosuppressive drugs include, for example: 1)alkylating agents such as nitrogen mustards (e.g., melphalan) andnitrosoureas (e.g., streptozocin); 2) antimetabolites such as folic acidantagonists (e.g., methotrexate), purine analogs (e.g., thioguanine),and pyrimidine analogs (e.g., gemcitabine); 3) cytotoxic antibioticssuch as anthracyclines (e.g., doxorubicin); 4) kinase inhibitors (e.g.,gefitinib); 5) mitotic inhibitors such as taxanes (e.g., paclitaxel) andvinca alkaloids (e.g., vinorelbine); 6) monoclonal antibodies (e.g.,rituximab); and 7) topoisomerase inhibitors (e.g., topotecan andetoposide). In addition, conditions resulting in a hypometabolic ratecan produce a mild-to-moderate hypoproliferative anemia. Among suchconditions are endocrine deficiency states. For example, anemia canoccur in Addison's disease, hypothyroidism, hyperparathyroidism, ormales who are castrated or treated with estrogen. In some embodiments,one or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure, optionally combined with an EPO receptoractivator, could be used to treat a hyperproliferative anemia.

Chronic kidney disease is sometimes associated with hypoproliferativeanemia, and the degree of the anemia varies in severity with the levelof renal impairment. Such anemia is primarily due to inadequateproduction of erythropoietin and reduced survival of red blood cells.Chronic kidney disease usually proceeds gradually over a period of yearsor decades to end-stage (Stage 5) disease, at which point dialysis orkidney transplantation is required for patient survival. Anemia oftendevelops early in this process and worsens as disease progresses. Theclinical consequences of anemia of kidney disease are well-documentedand include development of left ventricular hypertrophy, impairedcognitive function, reduced quality of life, and altered immune function[see, e.g., Levin et al. (1999) Am J Kidney Dis 27:347-354; Nissenson(1992) Am J Kidney Dis 20(Suppl 1):21-24; Revicki et al. (1995) Am JKidney Dis 25:548-554; Gafter et al., (1994) Kidney Int 45:224-231]. Insome embodiments, one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure, optionally combined with anEPO receptor activator, could be used to treat anemia associated withacute or chronic renal disease or failure.

Anemia resulting from acute blood loss of sufficient volume, such asfrom trauma or postpartum hemorrhage, is known as acute post-hemorrhagicanemia. Acute blood loss initially causes hypovolemia without anemiasince there is proportional depletion of RBCs along with other bloodconstituents. However, hypovolemia will rapidly trigger physiologicmechanisms that shift fluid from the extravascular to the vascularcompartment, which results in hemodilution and anemia. If chronic, bloodloss gradually depletes body iron stores and eventually leads to irondeficiency. In some embodiments, one or more TGF-beta superfamilyreceptor single-arm heteromultimer complexes of the disclosure,optionally combined with an EPO receptor activator, could be used totreat anemia resulting from acute blood loss.

Iron-deficiency anemia is the final stage in a graded progression ofincreasing iron deficiency which includes negative iron balance andiron-deficient erythropoiesis as intermediate stages. Iron deficiencycan result from increased iron demand, decreased iron intake, orincreased iron loss, as exemplified in conditions such as pregnancy,inadequate diet, intestinal malabsorption, acute or chronicinflammation, and acute or chronic blood loss. With mild-to-moderateanemia of this type, the bone marrow remains hypoproliferative, and RBCmorphology is largely normal; however, even mild anemia can result insome microcytic hypochromic RBCs, and the transition to severeiron-deficient anemia is accompanied by hyperproliferation of the bonemarrow and increasingly prevalent microcytic and hypochromic RBCs [see,e.g., Adamson (2008) Harrison's Principles of Internal Medicine, 17thed.; McGraw Hill, New York, pp 628-634]. Appropriate therapy foriron-deficiency anemia depends on its cause and severity, with oral ironpreparations, parenteral iron formulations, and RBC transfusion as majorconventional options. In some embodiments, one or more TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure, optionally combined with an EPO receptor activator, could beused to treat a chronic iron-deficiency.

Myelodysplastic syndrome (MDS) is a diverse collection of hematologicalconditions characterized by ineffective production of myeloid bloodcells and risk of transformation to acute myelogenous leukemia. In MDSpatients, blood stem cells do not mature into healthy red blood cells,white blood cells, or platelets. MDS disorders include, for example,refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, refractory cytopenia with multilineagedysplasia, and myelodysplastic syndrome associated with an isolated 5qchromosome abnormality. As these disorders manifest as irreversibledefects in both quantity and quality of hematopoietic cells, most MDSpatients are afflicted with chronic anemia. Therefore, MDS patientseventually require blood transfusions and/or treatment with growthfactors (e.g., erythropoietin or G-CSF) to increase red blood celllevels. However, many MDS patients develop side-effects due to frequencyof such therapies. For example, patients who receive frequent red bloodcell transfusion can exhibit tissue and organ damage from the buildup ofextra iron. Accordingly, one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure, may be used totreat patients having MDS. In certain embodiments, patients sufferingfrom MDS may be treated using one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure, optionally incombination with an EPO receptor activator. In other embodiments,patients suffering from MDS may be treated using a combination of one ormore TGF-beta superfamily receptor single-arm heteromultimer complexesof the disclosure and one or more additional therapeutic agents fortreating MDS including, for example, thalidomide, lenalidomide,azacitadine, decitabine, erythropoietins, deferoxamine, antithymocyteglobulin, and filgrastrim (G-CSF).

Originally distinguished from aplastic anemia, hemorrhage, or peripheralhemolysis on the basis of ferrokinetic studies [see, e.g., Ricketts etal. (1978) Clin Nucl Med 3:159-164], ineffective erythropoiesisdescribes a diverse group of anemias in which production of mature RBCsis less than would be expected given the number of erythroid precursors(erythroblasts) present in the bone marrow [Tanno et al. (2010) AdvHematol 2010:358283]. In such anemias, tissue hypoxia persists despiteelevated erythropoietin levels due to ineffective production of matureRBCs. A vicious cycle eventually develops in which elevatederythropoietin levels drive massive expansion of erythroblasts,potentially leading to splenomegaly (spleen enlargement) due toextramedullary erythropoiesis [see, e.g., Aizawa et al. (2003) Am JHematol 74:68-72], erythroblast-induced bone pathology [see, e.g., DiMatteo et al. (2008) J Biol Regul Homeost Agents 22:211-216], and tissueiron overload, even in the absence of therapeutic RBC transfusions [see,e.g., Pippard et al. (1979) Lancet 2:819-821]. Thus, by boostingerythropoietic effectiveness, one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the present disclosure may breakthe aforementioned cycle and thus alleviate not only the underlyinganemia but also the associated complications of elevated erythropoietinlevels, splenomegaly, bone pathology, and tissue iron overload. In someembodiments, one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the present disclosure can be used to treator prevent ineffective erythropoiesis, including anemia and elevated EPOlevels as well as complications such as splenomegaly,erythroblast-induced bone pathology, iron overload, and their attendantpathologies. With splenomegaly, such pathologies include thoracic orabdominal pain and reticuloendothelial hyperplasia. Extramedullaryhematopoiesis can occur not only in the spleen but potentially in othertissues in the form of extramedullary hematopoietic pseudotumors [see,e.g., Musallam et al. (2012) Cold Spring Harb Perspect Med 2:a013482].With erythroblast-induced bone pathology, attendant pathologies includelow bone mineral density, osteoporosis, and bone pain [see, e.g., Haidaret al. (2011) Bone 48:425-432]. With iron overload, attendantpathologies include hepcidin suppression and hyperabsorption of dietaryiron [see, e.g., Musallam et al. (2012) Blood Rev 26(Suppl 1):S16-S19],multiple endocrinopathies and liver fibrosis/cirrhosis [see, e.g.,Galanello et al. (2010) Orphanet J Rare Dis 5:11], and iron-overloadcardiomyopathy [Lekawanvijit et al., 2009, Can J Cardiol 25:213-218].

The most common causes of ineffective erythropoiesis are the thalassemiasyndromes, hereditary hemoglobinopathies in which imbalances in theproduction of intact alpha- and beta-hemoglobin chains lead to increasedapoptosis during erythroblast maturation [see, e.g., Schrier (2002) CurrOpin Hematol 9:123-126]. Thalassemias are collectively among the mostfrequent genetic disorders worldwide, with changing epidemiologicpatterns predicted to contribute to a growing public health problem inboth the U.S. and globally [Vichinsky (2005) Ann NY Acad Sci1054:18-24]. Thalassemia syndromes are named according to theirseverity. Thus, α-thalassemias include α-thalassemia minor (also knownas α-thalassemia trait; two affected α-globin genes), hemoglobin Hdisease (three affected α-globin genes), and α-thalassemia major (alsoknown as hydrops fetalis; four affected α-globin genes). β-Thalassemiasinclude β-thalassemia minor (also known as β-thalassemia trait; oneaffected β-globin gene), β-thalassemia intermedia (two affected β-globingenes), hemoglobin E thalassemia (two affected β-globin genes), andβ-thalassemia major (also known as Cooley's anemia; two affectedβ-globin genes resulting in a complete absence of β-globin protein).β-Thalassemia impacts multiple organs, is associated with considerablemorbidity and mortality, and currently requires life-long care. Althoughlife expectancy in patients with β-thalassemia has increased in recentyears due to use of regular blood transfusions in combination with ironchelation, iron overload resulting both from transfusions and fromexcessive gastrointestinal absorption of iron can cause seriouscomplications such as heart disease, thrombosis, hypogonadism,hypothyroidism, diabetes, osteoporosis, and osteopenia [see, e.g., Rundet al. (2005) N Engl J Med 353:1135-1146]. In certain embodiments, oneor more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure, optionally combined with an EPO receptoractivator, can be used to treat or prevent a thalassemia syndrome.

In some embodiments, one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure, optionallycombined with an EPO receptor activator, can be used for treatingdisorders of ineffective erythropoiesis besides thalassemia syndromes.Such disorders include siderblastic anemia (inherited or acquired);dyserythropoietic anemia (types I and II); sickle cell anemia;hereditary spherocytosis; pyruvate kinase deficiency; megaloblasticanemias, potentially caused by conditions such as folate deficiency (dueto congenital diseases, decreased intake, or increased requirements),cobalamin deficiency (due to congenital diseases, pernicious anemia,impaired absorption, pancreatic insufficiency, or decreased intake),certain drugs, or unexplained causes (congenital dyserythropoieticanemia, refractory megaloblastic anemia, or erythroleukemia);myelophthisic anemias including, for example, myelofibrosis (myeloidmetaplasia) and myelophthisis; congenital erythropoietic porphyria; andlead poisoning.

In certain embodiments, one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may be used incombination with supportive therapies for ineffective erythropoiesis.Such therapies include transfusion with either red blood cells or wholeblood to treat anemia. In chronic or hereditary anemias, normalmechanisms for iron homeostasis are overwhelmed by repeatedtransfusions, eventually leading to toxic and potentially fatalaccumulation of iron in vital tissues such as heart, liver, andendocrine glands. Thus, supportive therapies for patients chronicallyafflicted with ineffective erythropoiesis also include treatment withone or more iron-chelating molecules to promote iron excretion in theurine and/or stool and thereby prevent, or reverse, tissue iron overload[see, e.g., Hershko (2006) Haematologica 91:1307-1312; Cao et al.(2011), Pediatr Rep 3(2):e17]. Effective iron-chelating agents should beable to selectively bind and neutralize ferric iron, the oxidized formof non-transferrin bound iron which likely accounts for most irontoxicity through catalytic production of hydroxyl radicals and oxidationproducts [see, e.g., Esposito et al. (2003) Blood 102:2670-2677]. Theseagents are structurally diverse, but all possess oxygen or nitrogendonor atoms able to form neutralizing octahedral coordination complexeswith individual iron atoms in stoichiometries of 1:1 (hexadentateagents), 2:1 (tridentate), or 3:1 (bidentate) [Kalinowski et al. (2005)Pharmacol Rev 57:547-583]. In general, effective iron-chelating agentsalso are relatively low molecular weight (e.g., less than 700 daltons),with solubility in both water and lipids to enable access to affectedtissues. Specific examples of iron-chelating molecules includedeferoxamine, a hexadentate agent of bacterial origin requiring dailyparenteral administration, and the orally active synthetic agentsdeferiprone (bidentate) and deferasirox (tridentate). Combinationtherapy consisting of same-day administration of two iron-chelatingagents shows promise in patients unresponsive to chelation monotherapyand also in overcoming issues of poor patient compliance withdereroxamine alone [Cao et al. (2011) Pediatr Rep 3(2):e17; Galanello etal. (2010) Ann NY Acad Sci 1202:79-86].

As used herein, “in combination with” or “conjoint administration”refers to any form of administration such that the second therapy isstill effective in the body (e.g., the two compounds are simultaneouslyeffective in the patient, which may include synergistic effects of thetwo compounds). Effectiveness may not correlate to measurableconcentration of the agent in blood, serum, or plasma. For example, thedifferent therapeutic compounds can be administered either in the sameformulation or in separate formulations, either concomitantly orsequentially, and on different schedules. Thus, an individual whoreceives such treatment can benefit from a combined effect of differenttherapies. One or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional agents or supportive therapies. In general, each therapeuticagent will be administered at a dose and/or on a time scheduledetermined for that particular agent. The particular combination toemploy in a regimen will take into account compatibility of theantagonist of the present disclosure with the therapy and/or the desiredtherapeutic effect to be achieved.

In certain embodiments, one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may be used incombination with hepcidin or a hepcidin agonist for ineffectiveerythropoiesis. A circulating polypeptide produced mainly in the liver,hepcidin is considered a master regulator of iron metabolism by virtueof its ability to induce the degradation of ferroportin, an iron-exportprotein localized on absorptive enterocytes, hepatocytes, andmacrophages. Broadly speaking, hepcidin reduces availability ofextracellular iron, so hepcidin agonists may be beneficial in thetreatment of ineffective erythropoiesis [see, e.g., Nemeth (2010) AdvHematol 2010:750643]. This view is supported by beneficial effects ofincreased hepcidin expression in a mouse model of β-thalassemia[Gardenghi et al. (2010) J Clin Invest 120:4466-4477].

One or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure, optionally combined with an EPO receptoractivator, would also be appropriate for treating anemias of disorderedRBC maturation, which are characterized in part by undersized(microcytic), oversized (macrocytic), misshapen, or abnormally colored(hypochromic) RBCs.

In certain embodiments, the present disclosure provides methods oftreating or preventing anemia in an individual in need thereof byadministering to the individual a therapeutically effective amount ofone or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure and a EPO receptor activator. In certainembodiments, one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure may be used in combinationwith EPO receptor activators to reduce the required dose of theseactivators in patients that are susceptible to adverse effects of EPO.These methods may be used for therapeutic and prophylactic treatments ofa patient.

One or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure may be used in combination with EPO receptoractivators to achieve an increase in red blood cells, particularly atlower dose ranges of EPO receptor activators. This may be beneficial inreducing the known off-target effects and risks associated with highdoses of EPO receptor activators. The primary adverse effects of EPOinclude, for example, an excessive increase in the hematocrit orhemoglobin levels and polycythemia. Elevated hematocrit levels can leadto hypertension (more particularly aggravation of hypertension) andvascular thrombosis. Other adverse effects of EPO which have beenreported, some of which relate to hypertension, are headaches,influenza-like syndrome, obstruction of shunts, myocardial infarctionsand cerebral convulsions due to thrombosis, hypertensive encephalopathy,and red cell blood cell aplasia. See, e.g., Singibarti (1994) J. ClinInvestig 72(suppl 6), S36-S43; Horl et al. (2000) Nephrol DialTransplant 15(suppl 4), 51-56; Delanty et al. (1997) Neurology 49,686-689; and Bunn (2002) N Engl J Med 346(7), 522-523).

Provided that TGF-beta superfamily receptor single-arm heteromultimercomplexes of the present disclosure act by a different mechanism thanEPO, these antagonists may be useful for increasing red blood cell andhemoglobin levels in patients that do not respond well to EPO. Forexample, a TGF-beta superfamily receptor single-arm heteromultimercomplex of the present disclosure may be beneficial for a patient inwhich administration of a normal-to-increased dose of EPO (>300IU/kg/week) does not result in the increase of hemoglobin level up tothe target level. Patients with an inadequate EPO response are found inall types of anemia, but higher numbers of non-responders have beenobserved particularly frequently in patients with cancers and patientswith end-stage renal disease. An inadequate response to EPO can beeither constitutive (observed upon the first treatment with EPO) oracquired (observed upon repeated treatment with EPO).

In certain embodiments, the present disclosure provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure by measuring one or morehematologic parameters in the patient. The hematologic parameters may beused to evaluate appropriate dosing for a patient who is a candidate tobe treated with the antagonist of the present disclosure, to monitor thehematologic parameters during treatment, to evaluate whether to adjustthe dosage during treatment with one or more antagonist of thedisclosure, and/or to evaluate an appropriate maintenance dose of one ormore antagonists of the disclosure. If one or more of the hematologicparameters are outside the normal level, dosing with one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure may be reduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, usingart-recognized methods. Such parameters may be determined using a bloodsample from a patient. Increases in red blood cell levels, hemoglobinlevels, and/or hematocrit levels may cause increases in blood pressure.

In one embodiment, if one or more hematologic parameters are outside thenormal range or on the high side of normal in a patient who is acandidate to be treated with one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure, then onset ofadministration of the one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may be delayeduntil the hematologic parameters have returned to a normal or acceptablelevel either naturally or via therapeutic intervention. For example, ifa candidate patient is hypertensive or pre-hypertensive, then thepatient may be treated with a blood pressure lowering agent in order toreduce the patient's blood pressure. Any blood pressure lowering agentappropriate for the individual patient's condition may be usedincluding, for example, diuretics, adrenergic inhibitors (includingalpha blockers and beta blockers), vasodilators, calcium channelblockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensinII receptor blockers. Blood pressure may alternatively be treated usinga diet and exercise regimen. Similarly, if a candidate patient has ironstores that are lower than normal, or on the low side of normal, thenthe patient may be treated with an appropriate regimen of diet and/oriron supplements until the patient's iron stores have returned to anormal or acceptable level. For patients having higher than normal redblood cell levels and/or hemoglobin levels, then administration of theone or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure may be delayed until the levels havereturned to a normal or acceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range or on the high side of normal in a patient whois a candidate to be treated with one or more TGF-beta superfamilyreceptor single-arm heteromultimer complexes of the disclosure, then theonset of administration may not be delayed. However, the dosage amountor frequency of dosing of the one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may be set at anamount that would reduce the risk of an unacceptable increase in thehematologic parameters arising upon administration of the one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure. Alternatively, a therapeutic regimen may be developed forthe patient that combines one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure with a therapeuticagent that addresses the undesirable level of the hematologic parameter.For example, if the patient has elevated blood pressure, then atherapeutic regimen involving administration of one or more TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure and a blood pressure-lowering agent may be designed. For apatient having lower than desired iron stores, a therapeutic regimen ofone or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure and iron supplementation may be developed.

In one embodiment, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure and an appropriate dosingregimen established for that patient based on the baseline value(s).Alternatively, established baseline parameters based on a patient'smedical history could be used to inform an appropriate dosing regimenfor a patient. For example, if a healthy patient has an establishedbaseline blood pressure reading that is above the defined normal rangeit may not be necessary to bring the patient's blood pressure into therange that is considered normal for the general population prior totreatment with the one or more TGF-beta superfamily heteromultimercomplexes of the disclosure. A patient's baseline values for one or morehematologic parameters prior to treatment with one or more TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure may also be used as the relevant comparative values formonitoring any changes to the hematologic parameters during treatmentwith the one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with a one or more TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure. The hematologic parameters may be used to monitor thepatient during treatment and permit adjustment or termination of thedosing with the one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure or additional dosing withanother therapeutic agent. For example, if administration of one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure of the disclosure results in an increase in blood pressure,red blood cell level, or hemoglobin level, or a reduction in ironstores, then the dose of the one or more TGF-beta superfamily receptorsingle-arm heteromultimer complexes of the disclosure may be reduced inamount or frequency in order to decrease the effects of the one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure on the one or more hematologic parameters. If administrationof one or more TGF-beta superfamily receptor single-arm heteromultimercomplexes of the disclosure results in a change in one or morehematologic parameters that is adverse to the patient, then the dosingof the one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure may be terminated eithertemporarily, until the hematologic parameter(s) return to an acceptablelevel, or permanently. Similarly, if one or more hematologic parametersare not brought within an acceptable range after reducing the dose orfrequency of administration of the one or more TGF-beta superfamilyreceptor single-arm heteromultimer complexes of the disclosure, then thedosing may be terminated. As an alternative, or in addition to, reducingor terminating the dosing with the one or more TGF-beta superfamilyreceptor single-arm heteromultimer complexes of the disclosure, thepatient may be dosed with an additional therapeutic agent that addressesthe undesirable level in the hematologic parameter(s), such as, forexample, a blood pressure-lowering agent or an iron supplement. Forexample, if a patient being treated with one or more TGF-betasuperfamily receptor single-arm heteromultimer complexes of thedisclosure has elevated blood pressure, then dosing with the one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure may continue at the same level and a blood pressure-loweringagent is added to the treatment regimen, dosing with the one or moreTGF-beta superfamily receptor single-arm heteromultimer complexes of thedisclosure may be reduced (e.g., in amount and/or frequency) and a bloodpressure-lowering agent is added to the treatment regimen, or dosingwith the one or more TGF-beta superfamily receptor single-armheteromultimer complexes of the disclosure may be terminated and thepatient may be treated with a blood pressure-lowering agent.

6. Pharmaceutical Compositions

In certain aspects, TGF-beta superfamily receptor single-armheteromultimer complexes of the present disclosure can be administeredalone or as a component of a pharmaceutical formulation (also referredto as a therapeutic composition or pharmaceutical composition). Apharmaceutical formation refers to a preparation which is in such formas to permit the biological activity of an active ingredient (e.g., anagent of the present disclosure) contained therein to be effective andwhich contains no additional components which are unacceptably toxic toa subject to which the formulation would be administered. The subjectcompounds may be formulated for administration in any convenient way foruse in human or veterinary medicine. For example, one or more agents ofthe present disclosure may be formulated with a pharmaceuticallyacceptable carrier. A pharmaceutically acceptable carrier refers to aningredient in a pharmaceutical formulation, other than an activeingredient, which is generally nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, and/or preservative. In general, pharmaceutical formulationsfor use in the present disclosure are in a pyrogen-free,physiologically-acceptable form when administered to a subject.Therapeutically useful agents other than those described herein, whichmay optionally be included in the formulation as described above, may beadministered in combination with the subject agents in the methods ofthe present disclosure.

In certain embodiments, compositions will be administered parenterally[e.g., by intravenous (I. V.) injection, intraarterial injection,intraosseous injection, intramuscular injection, intrathecal injection,subcutaneous injection, or intradermal injection]. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore agents of the disclosure in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use. Injectable solutions or dispersions maycontain antioxidants, buffers, bacteriostats, suspending agents,thickening agents, or solutes which render the formulation isotonic withthe blood of the intended recipient. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalformulations of the present disclosure include water, ethanol, polyols(e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetableoils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate),and suitable mixtures thereof. Proper fluidity can be maintained, forexample, by the use of coating materials (e.g., lecithin), by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In some embodiments, a therapeutic method of the present disclosureincludes administering the pharmaceutical composition systemically, orlocally, from an implant or device. Further, the pharmaceuticalcomposition may be encapsulated or injected in a form for delivery to atarget tissue site (e.g., bone marrow or muscle). In certainembodiments, compositions of the present disclosure may include a matrixcapable of delivering one or more of the agents of the presentdisclosure to a target tissue site (e.g., bone marrow or muscle),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of one or more agents of the present disclosure. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material may be based on one or more of:biocompatibility, biodegradability, mechanical properties, cosmeticappearance, and interface properties. The particular application of thesubject compositions will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid, and polyanhydrides. Other potential materials are biodegradableand biologically well-defined including, for example, bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenon-biodegradable and chemically defined including, for example,sintered hydroxyapatite, bioglass, aluminates, or other ceramics.Matrices may be comprised of combinations of any of the above mentionedtypes of material including, for example, polylactic acid andhydroxyapatite or collagen and tricalciumphosphate. The bioceramics maybe altered in composition (e.g., calcium-aluminate-phosphate) andprocessing to alter one or more of pore size, particle size, particleshape, and biodegradability.

In certain embodiments, pharmaceutical compositions of the presentdisclosure can be administered topically. “Topical application” or“topically” means contact of the pharmaceutical composition with bodysurfaces including, for example, the skin, wound sites, and mucousmembranes. The topical pharmaceutical compositions can have variousapplication forms and typically comprises a drug-containing layer, whichis adapted to be placed near to or in direct contact with the tissueupon topically administering the composition. Pharmaceuticalcompositions suitable for topical administration may comprise one ormore TGFβ superfamily receptor single-arm heteromultimer complexes ofthe disclosure in combination formulated as a liquid, a gel, a cream, alotion, an ointment, a foam, a paste, a putty, a semi-solid, or a solid.Compositions in the liquid, gel, cream, lotion, ointment, foam, paste,or putty form can be applied by spreading, spraying, smearing, dabbingor rolling the composition on the target tissue. The compositions alsomay be impregnated into sterile dressings, transdermal patches,plasters, and bandages. Compositions of the putty, semi-solid or solidforms may be deformable. They may be elastic or non-elastic (e.g.,flexible or rigid). In certain aspects, the composition forms part of acomposite and can include fibers, particulates, or multiple layers withthe same or different compositions.

Topical compositions in the liquid form may include pharmaceuticallyacceptable solutions, emulsions, microemulsions, and suspensions. Inaddition to the active ingredient(s), the liquid dosage form may containan inert diluent commonly used in the art including, for example, wateror other solvent, a solubilizing agent and/or emulsifier [e.g., ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, anoil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fattyacid ester of sorbitan, and mixtures thereof].

Topical gel, cream, lotion, ointment, semi-solid or solid compositionsmay include one or more thickening agents, such as a polysaccharide,synthetic polymer or protein-based polymer. In one embodiment of theinvention, the gelling agent herein is one that is suitably nontoxic andgives the desired viscosity. The thickening agents may include polymers,copolymers, and monomers of: vinylpyrrolidones, methacrylamides,acrylamides N-vinylimidazoles, carboxy vinyls, vinyl esters, vinylethers, silicones, polyethyleneoxides, polyethyleneglycols,vinylalcohols, sodium acrylates, acrylates, maleic acids,NN-dimethylacrylamides, diacetone acrylamides, acrylamides, acryloylmorpholine, pluronic, collagens, polyacrylamides, polyacrylates,polyvinyl alcohols, polyvinylenes, polyvinyl silicates, polyacrylatessubstituted with a sugar (e.g., sucrose, glucose, glucosamines,galactose, trehalose, mannose, or lactose), acylamidopropane sulfonicacids, tetramethoxyorthosilicates, methyltrimethoxyorthosilicates,tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propyleneglycol, glycerine, polysaccharides, alginates, dextrans, cyclodextrin,celluloses, modified celluloses, oxidized celluloses, chitosans,chitins, guars, carrageenans, hyaluronic acids, inulin, starches,modified starches, agarose, methylcelluloses, plant gums, hylaronans,hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses,hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins,low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graftcopolymers, starch sodium polyacrylate, hydroxyethyl methacrylates,hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethylmethacrylates, polystyrenes, polyurethanes, polyalkanoates, polylacticacids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS(2-acrylamido-2-methyl-1-propanesulfonic acid), SEM(sulfoethylmethacrylate), SPM (sulfopropyl methacrylate), SPA(sulfopropyl acrylate),N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine,methacryllic acid amidopropyl-dimethyl ammonium sulfobetaine, SPI(itaconic acid-bis(1-propyl sulfonizacid-3) ester di-potassium salt),itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid),beta-carboxyethyl acrylate (acrylic acid dimers), and maleicanhydride-methylvinyl ether polymers, derivatives thereof, saltsthereof, acids thereof, and combinations thereof. In certainembodiments, pharmaceutical compositions of present disclosure can beadministered orally, for example, in the form of capsules, cachets,pills, tablets, lozenges (using a flavored basis such as sucrose andacacia or tragacanth), powders, granules, a solution or a suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, or an elixir or syrup, or pastille (using an inert base, suchas gelatin and glycerin, or sucrose and acacia), and/or a mouth wash,each containing a predetermined amount of a compound of the presentdisclosure and optionally one or more other active ingredients. Acompound of the present disclosure and optionally one or more otheractive ingredients may also be administered as a bolus, electuary, orpaste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, and granules), one or more compounds of thepresent disclosure may be mixed with one or more pharmaceuticallyacceptable carriers including, for example, sodium citrate, dicalciumphosphate, a filler or extender (e.g., a starch, lactose, sucrose,glucose, mannitol, and silicic acid), a binder (e.g.carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone,sucrose, and acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, and sodium carbonate), a solution retardingagent (e.g. paraffin), an absorption accelerator (e.g. a quaternaryammonium compound), a wetting agent (e.g., cetyl alcohol and glycerolmonostearate), an absorbent (e.g., kaolin and bentonite clay), alubricant (e.g., a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), a coloring agent, andmixtures thereof. In the case of capsules, tablets, and pills, thepharmaceutical formulation (composition) may also comprise a bufferingagent. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using one or moreexcipients including, e.g., lactose or a milk sugar as well as a highmolecular-weight polyethylene glycol.

Liquid dosage forms for oral administration of the pharmaceuticalcomposition may include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient(s), the liquid dosage form may contain an inertdiluent commonly used in the art including, for example, water or othersolvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oil),glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acidester of sorbitan, and mixtures thereof]. Besides inert diluents, theoral formulation can also include an adjuvant including, for example, awetting agent, an emulsifying and suspending agent, a sweetening agent,a flavoring agent, a coloring agent, a perfuming agent, a preservativeagent, and combinations thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents including, for example, an ethoxylated isostearyl alcohol,polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, tragacanth, andcombinations thereof.

Prevention of the action and/or growth of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agentsincluding, for example, paraben, chlorobutanol, and phenol sorbic acid.

In certain embodiments, it may be desirable to include an isotonic agentincluding, for example, a sugar or sodium chloride into thecompositions. In addition, prolonged absorption of an injectablepharmaceutical form may be brought about by the inclusion of an agentthat delay absorption including, for example, aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the one or more of the agents of the present disclosure. In the caseof a TGF-beta superfamily receptor single-arm heteromultimer complexthat promotes red blood cell formation, various factors may include, butare not limited to, the patient's red blood cell count, hemoglobinlevel, the desired target red blood cell count, the patient's age, thepatient's sex, the patient's diet, the severity of any disease that maybe contributing to a depressed red blood cell level, the time ofadministration, and other clinical factors. The addition of other knownactive agents to the final composition may also affect the dosage.Progress can be monitored by periodic assessment of one or more of redblood cell levels, hemoglobin levels, reticulocyte levels, and otherindicators of the hematopoietic process.

In certain embodiments, the present disclosure also provides genetherapy for the in vivo production of one or more of the agents of thepresent disclosure. Such therapy would achieve its therapeutic effect byintroduction of the agent sequences into cells or tissues having one ormore of the disorders as listed above. Delivery of the agent sequencescan be achieved, for example, by using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Preferredtherapeutic delivery of one or more of agent sequences of the disclosureis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus(e.g., a retrovirus). The retroviral vector may be a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target-specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing one or more of theagents of the present disclosure.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes (gag, pol, and env),by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for one or more of the agents of thepresent disclosure is a colloidal dispersion system. Colloidaldispersion systems include, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Incertain embodiments, the preferred colloidal system of this disclosureis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. RNA, DNA, and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form. See, e.g., Fraley, et al. (1981)Trends Biochem. Sci., 6:77. Methods for efficient gene transfer using aliposome vehicle are known in the art. See, e.g., Mannino, et al. (1988)Biotechniques, 6:682, 1988.

The composition of the liposome is usually a combination ofphospholipids, which may include a steroid (e.g. cholesterol). Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Other phospholipids or other lipidsmay also be used including, for example a phosphatidyl compound (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, a sphingolipid, a cerebroside, and aganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine,and distearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1. Generation and Characterization of a Single-Arm ActRIIB-FcHeterodimer

Applicants constructed a soluble single-arm ActRIIB-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman ActRIIB was fused to a separate Fc domain with a linker positionedbetween the extracellular domain and this second Fc domain. Theindividual constructs are referred to as monomeric Fc polypeptide andActRIIB-Fc fusion polypeptide, respectively, and the sequences for eachare provided below.

A methodology for promoting formation of ActRIIB-Fc:Fc heteromericcomplexes rather than ActRIIB-Fc:ActRIM-Fc or Fc:Fc homodimericcomplexes is to introduce alterations in the amino acid sequence of theFc domains to guide the formation of asymmetric heteromeric complexes.Many different approaches to making asymmetric interaction pairs usingFc domains are described in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and monomeric Fcpolypeptide sequences of SEQ ID NOs: 104-106 and 137-139, respectively,one Fc domain is altered to introduce cationic amino acids at theinteraction face, while the other Fc domain is altered to introduceanionic amino acids at the interaction face. The ActRIIB-Fc fusionpolypeptide and monomeric Fc polypeptide each employ the tissueplasminogen activator (TPA) leader:

(SEQ ID NO: 100) MDAMKRGLCCVLLLCGAVFVSP.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 104) is shown below:

(SEQ ID NO: 104) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNANWELERTNQS 51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151 PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201 DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP 251 APIEKTISKA KGQPREPQVY TLPPSRKEMTKNQVSLTCLV KGFYPSDIAV 301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH 351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of the ActRIIB-Fc:Fc heterodimer rather than either of thepossible homodimeric complexes (ActRIIB-Fc:ActRIIB-Fc or Fc:Fc), twoamino acid substitutions (replacing acidic amino acids with lysine) canbe introduced into the Fc domain of the ActRIM fusion protein asindicated by double underline above. The amino acid sequence of SEQ IDNO: 104 may optionally be provided with the C-terminal lysine (K)removed.

This ActRIIB-Fc fusion polypeptide is encoded by the following nucleicacid sequence (SEQ ID NO: 105):

(SEQ ID NO: 105) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC 151 GGCCTGGAGCGCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC 201 CTCCTGGCGC AACAGCTCTGGCACCATCGA GCTCGTGAAG AAGGGCTGCT 251 GGCTAGATGA CTTCAACTGC TACGATAGGCAGGAGTGTGT GGCCACTGAG 301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACTTCTGCAACGA 351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC 451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 501 ACCCAAGGACACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 551 TGGTGGACGT GAGCCACGAAGACCCTGAGG TCAAGTTCAA CTGGTACGTG 601 GACGGCGTGG AGGTGCATAA TGCCAAGACAAAGCCGCGGG AGGAGCAGTA 651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTGCACCAGGACT 701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 801ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG 851 TCAGCCTGACCTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG 901 GAGTGGGAGA GCAATGGGCAGCCGGAGAAC AACTACAAGA CCACGCCTCC 951 CGTGCTGAAG TCCGACGGCT CCTTCTTCCTCTATAGCAAG CTCACCGTGG 1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTCCGTGATGCAT 1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG1101 TAAA

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 106) is as followsand may optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 106) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHCYASWRNSSGT 51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151 RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201 VLTVLHQDWL NGKEYKCKVSNKALPAPIEK TISKAKGQPR EPQVYTLPPS 251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLKSDGSF 301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGK

The complementary human G1Fc polypeptide (SEQ ID NO: 137) employs theTPA leader and is as follows:

(SEQ ID NO: 137) 1 MDAMKRGLCC VLLLCGAVFV SPGA SNTKVD KRVTGGGTHTCPPCPAPELL 51 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 101NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 151 ISKAKGQPREPQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG 201 QPENNYDTTP PVLDSDGSFFLYSDLTVDKS RWQQGNVFSC SVMHEALHNH 251 YTQKSLSLSP GK

The leader sequence is underlined, and an optional N-terminal extensionof the Fc polypeptide is indicated by double underline. To promoteformation of the ActRIIB-Fc:Fc heterodimer rather than either of thepossible homodimeric complexes, two amino acid substitutions (replacinglysines with anionic residues) can be introduced into the monomeric Fcpolypeptide as indicated by double underline above. The amino acidsequence of SEQ ID NO: 137 may optionally be provided with theC-terminal lysine removed.

This complementary Fc polypeptide is encoded by the following nucleicacid (SEQ ID NO: 138).

(SEQ ID NO: 138) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCAGCAACAC CAAGGTGGAC AAGAGAGTTA 101CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG 151 GGGGGACCGTCAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT 201 GATCTCCCGG ACCCCTGAGGTCACATGCGT GGTGGTGGAC GTGAGCCACG 251 AAGACCCTGA GGTCAAGTTC AACTGGTACGTGGACGGCGT GGAGGTGCAT 301 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCACGTACCGTGT 351 GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT401 ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC 451ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC 501 CCCATCCCGGGAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG 551 TCAAAGGCTT CTATCCCAGCGACATCGCCG TGGAGTGGGA GAGCAATGGG 601 CAGCCGGAGA ACAACTACGA CACCACGCCTCCCGTGCTGG ACTCCGACGG 651 CTCCTTCTTC CTCTATAGCG ACCTCACCGT GGACAAGAGCAGGTGGCAGC 701 AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC751 TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

The sequence of the mature monomeric Fc polypeptide is as follows (SEQID NO: 139) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 139) 1 SNTKVDKRVT GGGTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV 51 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 101HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 151 KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYDTTPPVLD SDGSFFLYSD 201 LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK

The ActRIIB-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 106 and SEQ ID NO: 139, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ActRIIB-Fc:Fc.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion polypeptides, the Fc domains are altered tointroduce complementary hydrophobic interactions and an additionalintermolecular disulfide bond, as illustrated in the ActRIIB-Fc andmonomeric Fc polypeptide sequences of SEQ ID NOs: 403-404 and 425-426,respectively.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 403) employs the TPAleader and is shown below:

(SEQ ID NO: 403) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNANWELERTNQS 51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151 PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201 DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP 251 APIEKTISKA KGQPREPQVY TLPPCREEMTKNQVSLWCLV KGFYPSDIAV 301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH 351 EALHNHYTQK SLSLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ActRIIB-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a trytophan) can be introduced intothe Fc domain of the fusion protein as indicated by double underlineabove. The amino acid sequence of SEQ ID NO: 403 may optionally beprovided with the C-terminal lysine removed.

The mature ActRIM-Fc fusion polypeptide is as follows:

(SEQ ID NO: 404) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHCYASWRNSSGT 51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151 RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201 VLTVLHQDWL NGKEYKCKVSNKALPAPIEK TISKAKGQPR EPQVYTLPPC 251 REEMTKNQVS LWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSF 301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGK

The complementary form of monomeric Fc polypeptide (SEQ ID NO: 425) usesthe TPA leader and is as follows.

(SEQ ID NO: 425) 1 MDAMKRGLCC VLLLCGAVFV SPGA SNTKVD KRVTGGGTHTCPPCPAPELL 51 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 101NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 151 ISKAKGQPREPQVCTLPPSR EEMTKNQVSL SCAVKGFYPS DIAVEWESNG 201 QPENNYKTTP PVLDSDGSFFLVSKLTVDKS RWQQGNVFSC SVMHEALHNH 251 YTQKSLSLSP GK

The leader sequence is underlined, and an optional N-terminal extensionof the Fc polypeptide is indicated by double underline. To promoteformation of the ActRIIB-Fc:Fc heterodimer rather than either of thepossible homodimeric complexes, four amino acid substitutions can beintroduced into the monomeric Fc polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 425 mayoptionally be provided with the C-terminal lysine removed.

The mature monomeric Fc polypeptide sequence (SEQ ID NO: 426) is asfollows and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 426) 1 SNTKVDKRVT GGGTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV 51 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 101HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVC TLPPSREEMT 151 KNQVSLSCAVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK 201 LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK

The ActRIIB-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 404 and SEQ ID NO: 426, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ActRIIB-Fc:Fc.

Purification of various ActRIM-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm ActRIIB-Fc heterodimeric complex describedabove with that of ActRIIB-Fc homodimeric complex. Single-arm ActRIIB-Fcand homodimeric ActRIIB-Fc were independently captured onto the systemusing an anti-Fc antibody. Ligands were injected and allowed to flowover the captured receptor protein. Results are summarized in the tablebelow, in which ligand off-rates (k_(d)) typically associated with themost effective ligand traps are denoted in bold.

Ligand binding by single-arm ActRIIB-Fc compared to ActRIIB-Fc homodimerActRIIB-Fc homodimer Single-arm ActRIIB-Fc k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) Ligand (l/Ms) (l/s) (pM) (l/Ms) (l/s) (pM) Activin A 1.2 × 10⁷ 2.3× 10 ⁻⁴ 19 3.0 × 10⁷ 3.0 × 10⁻³ 99 Activin B 5.1 × 10⁶ 1.0 × 10 ⁻⁴ 203.5 × 10⁶ 4.2 × 10 ⁻⁴ 120 BMP6 3.2 × 10⁷ 6.8 × 10⁻³ 210 4.2 × 10⁷ 2.9 ×10⁻² 690 BMP9 1.4 × 10⁷ 1.1 × 10⁻³ 78 No binding BMP10 2.3 × 10⁷ 2.6 ×10 ⁻⁴ 11 8.0 × 10⁷ 9.7 × 10⁻³ 120 GDF3 1.4 × 10⁶ 2.2 × 10⁻³ 1500 1.1 ×10⁶ 1.3 × 10⁻² 12000 GDF8 8.3 × 10⁵ 2.3 × 10 ⁻⁴ 280 3.5 × 10⁶ 1.0 × 10⁻³290 GDF11 5.0 × 10⁷ 1.1 × 10 ⁻⁴ 2 3.6 × 10⁷ 7.2 × 10 ⁻⁴ 20

These comparative binding data demonstrate that single-arm ActRIIB-Fchas greater ligand selectivity than homodimeric ActRIIB-Fc. WhereasActRIIB-Fc homodimer binds strongly to five important ligands (seecluster of activin A, activin B, BMP10, GDF8, and GDF11 in FIG. 6),single-arm ActRIIB-Fc discriminates more readily among these ligands.Thus, single-arm ActRIIB-Fc binds strongly to activin B and GDF11 andwith intermediate strength to GDF8 and activin A. In further contrast toActRIIB-Fc homodimer, single-arm ActRIIB-Fc displays only weak bindingto BMP10 and no binding to BMP9. See FIG. 6.

These results indicate that single-arm ActRIIB-Fc is a more selectiveantagonist than ActRIIB-Fc homodimer. Accordingly, single-arm ActRIIB-Fcwill be more useful than ActRIIB-Fc homodimer in certain applicationswhere such selective antagonism is advantageous. Examples includetherapeutic applications where it is desirable to retain antagonism ofone or more of activin A, activin B, GDF8, and GDF11 but minimizeantagonism of one or more of BMP9, BMP10, BMP6, and GDF3. Selectiveinhibition of ligands in the former group would be particularlyadvantageous therapeutically because they constitute a subfamily whichtends to differ functionally from the latter group and its associatedset of clinical conditions.

Example 2. Generation and Characterization of a Single-Arm ALK3-FcHeterodimer

Applicants constructed a soluble single-arm ALK3-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman ALK3 was fused to a separate Fc domain with a linker positionedbetween the extracellular domain and this second Fc domain. Theindividual constructs are referred to as monomeric Fc polypeptide andALK3-Fc fusion polypeptide, respectively, and the sequences for each areprovided below.

Formation of a single-arm ALK3-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theALK3-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 122-124and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK3-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 122) 1 MDAMKRGLCC VLLLCGAVFV SPGAQNLDSM LHGTGMKSDSDQKKSENGVT 51 LAPEDTLPFL KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG 101CMKYEGSDFQ CKDSPKAQLR RTIECCRTNL CNQYLQPTLP PVVIGPFFDG 151 SIRTGGGTHTCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 201 VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 251 GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL 301 TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFFLYSDLTVDKS 351 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The leader and linker sequences are underlined. To promote formation ofthe ALK3-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK3-Fc:ALK3-Fc or Fc:Fc, two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion protein as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 122 mayoptionally be provided with a lysine added at the C-terminus.

This ALK3-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 123).

(SEQ ID NO: 123) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCCAGAATCT GGATAGTATG CTTCATGGCA 101CTGGGATGAA ATCAGACTCC GACCAGAAAA AGTCAGAAAA TGGAGTAACC 151 TTAGCACCAGAGGATACCTT GCCTTTTTTA AAGTGCTATT GCTCAGGGCA 201 CTGTCCAGAT GATGCTATTAATAACACATG CATAACTAAT GGACATTGCT 251 TTGCCATCAT AGAAGAAGAT GACCAGGGAGAAACCACATT AGCTTCAGGG 301 TGTATGAAAT ATGAAGGATC TGATTTTCAG TGCAAAGATTCTCCAAAAGC 351 CCAGCTACGC CGGACAATAG AATGTTGTCG GACCAATTTA TGTAACCAGT401 ATTTGCAACC CACACTGCCC CCTGTTGTCA TAGGTCCGTT TTTTGATGGC 451AGCATTCGAA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC 501 TGAACTCCTGGGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 551 ACACCCTCAT GATCTCCCGGACCCCTGAGG TCACATGCGT GGTGGTGGAC 601 GTGAGCCACG AAGACCCTGA GGTCAAGTTCAACTGGTACG TGGACGGCGT 651 GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAGTACAACAGCA 701 CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT751 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 801CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 851 ACACCCTGCCCCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 901 ACCTGCCTGG TCAAAGGCTTCTATCCCAGC GACATCGCCG TGGAGTGGGA 951 GAGCAATGGG CAGCCGGAGA ACAACTACGACACCACGCCT CCCGTGCTGG 1001 ACTCCGACGG CTCCTTCTTC CTCTATAGCG ACCTCACCGTGGACAAGAGC 1051 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT1101 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGT

The mature ALK3-Fc fusion polypeptide sequence is as follows (SEQ ID NO:124) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 124) 1 GAQNLDSMLH GTGMKSDSDQ KKSENGVTLA PEDTLPFLKCYCSGHCPDDA 51 INNTCITNGH CFAIIEEDDQ GETTLASGCM KYEGSDFQCK DSPKAQLRRT 101IECCRTNLCN QYLQPTLPPV VIGPFFDGSI RTGGGTHTCP PCPAPELLGG 151 PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA 201 KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 251 KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQP 301 ENNYDTTPPV LDSDGSFFLY SDLTVDKSRW QQGNVFSCSVMHEALHNHYT 351 QKSLSLSPG

The complementary human G1Fc polypeptide (SEQ ID NO: 140) employs theTPA leader and is as follows:

(SEQ ID NO: 140) 1 MDAMKRGLCC VLLLCGAVFV SPGA SNTKVD KRVTGGGTHTCPPCPAPELL 51 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 101NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 151 ISKAKGQPREPQVYTLPPSR KEMTKNQVSL TCLVKGFYPS DIAVEWESNG 201 QPENNYKTTP PVLKSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH 251 YTQKSLSLSP GK

The leader sequence is underlined, and an optional N-terminal extensionof the Fc polypeptide is indicated by double underline. To promoteformation of the ALK3-Fc:Fc heterodimer rather than either of thepossible homodimeric complexes, two amino acid substitutions (replacinganionic residues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated by double underline above. The amino acidsequence of SEQ ID NO: 140 may optionally be provided with theC-terminal lysine removed.

This complementary Fc polypeptide is encoded by the following nucleicacid (SEQ ID NO: 141).

(SEQ ID NO: 141) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCAGCAACAC CAAGGTGGAC AAGAGAGTTA 101CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG 151 GGGGGACCGTCAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT 201 GATCTCCCGG ACCCCTGAGGTCACATGCGT GGTGGTGGAC GTGAGCCACG 251 AAGACCCTGA GGTCAAGTTC AACTGGTACGTGGACGGCGT GGAGGTGCAT 301 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCACGTACCGTGT 351 GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT401 ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC 451ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC 501 CCCATCCCGGAAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG 551 TCAAAGGCTT CTATCCCAGCGACATCGCCG TGGAGTGGGA GAGCAATGGG 601 CAGCCGGAGA ACAACTACAA GACCACGCCTCCCGTGCTGA AGTCCGACGG 651 CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGCAGGTGGCAGC 701 AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC751 TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

The sequence of the mature monomeric Fc polypeptide is as follows (SEQID NO: 142) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 142) 1 SNTKVDKRVT GGGTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV 51 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 101HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRKEMT 151 KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLK SDGSFFLYSK 201 LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK

The ALK3-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 124 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK3-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK3-Fcand Fc polypeptide sequences of SEQ ID NOs: 415-416 and 427-428,respectively.

The ALK3-Fc fusion polypeptide (SEQ ID NO: 415) uses the TPA leader andis as follows:

(SEQ ID NO: 415) 1 MDAMKRGLCC VLLLCGAVFV SPGAQNLDSM LHGTGMKSDSDQKKSENGVT 51 LAPEDTLPFL KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG 101CMKYEGSDFQ CKDSPKAQLR RTIECCRTNL CNQYLQPTLP PVVIGPFFDG 151 SIRTGGGTHTCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 201 VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 251 GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVCTLPPSR EEMTKNQVSL 301 SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLVSKLTVDKS 351 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

The leader sequence and linker are underlined. To promote formation ofthe ALK3-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK3 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 415 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK3-Fc fusion polypeptide (SEQ ID NO: 416) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 416) 1 GAQNLDSMLH GTGMKSDSDQ KKSENGVTLA PEDTLPFLKCYCSGHCPDDA 51 INNTCITNGH CFAIIEEDDQ GETTLASGCM KYEGSDFQCK DSPKAQLRRT 101IECCRTNLCN QYLQPTLPPV VIGPFFDGSI RTGGGTHTCP PCPAPELLGG 151 PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA 201 KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 251 KAKGQPREPQ VCTLPPSREE MTKNQVSLSCAVKGFYPSDI AVEWESNGQP 301 ENNYKTTPPV LDSDGSFFLV SKLTVDKSRW QQGNVFSCSVMHEALHNHYT 351 QKSLSLSPGK

The complementary form of monomeric G1Fc polypeptide (SEQ ID NO: 427)employs the TPA leader and is as follows:

(SEQ ID NO: 427) 1 MDAMKRGLCC VLLLCGAVFV SPGA SNTKVD KRVTGGGTHTCPPCPAPELL 51 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 101NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 151 ISKAKGQPREPQVYTLPPCR EEMTKNQVSL WCLVKGFYPS DIAVEWESNG 201 QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH 251 YTQKSLSLSP GK

The leader sequence is underlined, and an optional N-terminal extensionof the Fc polypeptide is indicated by double underline. To promoteformation of the ALK3-Fc:Fc heterodimer rather than either of thepossible homodimeric complexes, two amino acid substitutions (replacinga serine with a cysteine and a threonine with a tryptophan) can beintroduced into the monomeric Fc polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 427 mayoptionally be provided with the C-terminal lysine removed.

The sequence of the mature monomeric Fc polypeptide is as follows (SEQID NO: 428) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 428) 1 SNTKVDKRVT GGGTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV 51 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 101HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCREEMT 151 KNQVSLWCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK 201 LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK

The ALK3-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 416 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericcomplex comprising ALK3-Fc:Fc.

Purification of various ALK3-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm ALK3-Fc heterodimeric complex describedabove with that of an ALK3-Fc homodimeric complex. The single-armALK3-Fc and homodimeric ALK3-Fc were independently captured onto thesystem using an anti-Fc antibody. Ligands were injected and allowed toflow over the captured receptor protein. Results are summarized in thetable below, in which ligand off-rates (k_(d)) typically associated withthe most effective ligand traps are denoted in bold.

Ligand binding of single-arm ALK3-Fc compared to ALK3-Fc homodimerALK3-Fc homodimer Single-arm ALK3-Fc k_(a) k_(d) K_(D) k_(a) k_(d) K_(D)Ligand (l/Ms) (l/s) (pM) (l/Ms) (l/s) (pM) Activin A No binding Nobinding Activin B No binding No binding Activin No binding No binding ABActivin No binding No binding AC BMP2 6.8 × 10⁵ 8.9 × 10 ⁻⁵ 130 7.9 ×10⁵ 2.5 × 10 ⁻⁴ 310 BMP4 3.0 × 10⁵ 5.3 × 10 ⁻⁵ 178 4.9 × 10⁵ 4.6 × 10 ⁻⁵93 BMP5 2.9 × 10⁴ 2.0 × 10⁻³ 70000 1.2 × 10⁵ 5.3 × 10⁻³ 45000 BMP6 1.4 ×10⁵ 4.9 × 10⁻³ 35000 No binding BMP7 1.2 × 10⁶ 1.8 × 10⁻² 15000 Nobinding BMP10 No binding No binding GDF5 4.8 × 10⁵ 1.1 × 10⁻² 22000 Nobinding GDF6 3.4 × 10⁴ 1.3 × 10⁻³ 40000 No binding GDF7 2.2 × 10⁵ 2.7 ×10⁻³ 12000 4.6 × 10⁵ 1.0 × 10⁻² 22000 GDF8 No binding No binding GDF11No binding No binding

These comparative data indicate that single-arm ALK3-Fc has greaterligand selectivity than homodimeric ALK3-Fc. Whereas single-arm ALK3-Fcheterodimer retains the exceptionally tight binding to BMP4 observedwith ALK3-Fc homodimer, it exhibits reduced strength of binding to BMP2and therefore discriminates better between BMP4 and BMP2 (still a strongbinder) than does ALK3-Fc homodimer. Single-arm ALK3-Fc alsodiscriminates better among BMP5 (intermediate binding), GDF7 (weakbinding), and GDF6 (no binding) compared to ALK3-Fc homodimer, whichbinds these three ligands with very similar strength (all intermediate).See FIG. 7. Unlike constructs disclosed in Example 1, neither single-armALK3-Fc nor homodimeric ALK3-Fc binds activins, GDF8, GDF11, or BMP10.

These results therefore indicate that single-arm ALK3-Fc is a moreselective antagonist of BMP4 than is ALK3-Fc homodimer. Single-armALK3-Fc can be expected to antagonize BMP4 in a more targetedmanner—with reduced effects from concurrent antagonism of BMP2 or BMP5and especially GDF6 or GDF7—compared to ALK3-Fc homodimer. Accordingly,single-arm ALK3-Fc will be more useful than ALK3-Fc homodimer in certainapplications where such selective antagonism is advantageous. Examplesinclude therapeutic applications where it is desirable to retainantagonism of one or more of BMP4, BMP2, and potentially BMP5 butminimize antagonism of one or more of BMP6, GDF6, and GDF7.

Example 3. Generation and Characterization of a Single-Arm ActRIIA-FcHeterodimer

Applicants constructed a soluble single-arm ActRIIA-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman ActRIIA was fused to a separate Fc domain with a linker positionedbetween the extracellular domain and this second Fc domain. Theindividual constructs are referred to as monomeric Fc polypeptide andActRIIA-Fc fusion polypeptide, respectively, and the sequences for eachare provided below.

Formation of a single-arm ActRIIA-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIM-Fcheterodimer in Example 1. In a first approach, illustrated in theActRIIA-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 101-103and 137-139, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ActRIIA-Fc fusion polypeptide employs the TPA leader and is asfollows:

(SEQ ID NO: 101) 1 MDAMKRGLCC VLLLCGAVFV SPGAAILGRS ETQECLFFNANWEKDRTNQT 51 GVEPCYGDKD KRRHCFATWK NISGSIEIVK QGCWLDDINC YDRTDCVEKK 101DSPEVYFCCC EGNMCNEKFS YFPEMEVTQP TSNPVTPKPP TGGGTHTCPP 151 CPAPELLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY 201 VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL 251 PAPIEKTISK AKGQPREPQV YTLPPSRKEMTKNQVSLTCL VKGFYPSDIA 301 VEWESNGQPE NNYKTTPPVL KSDGSFFLYS KLTVDKSRWQQGNVFSCSVM 351 HEALHNHYTQ KSLSLSPGK

The leader and linker sequences are underlined. To promote formation ofthe ActRIIA-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ActRIIA-Fc:ActRIIA-Fc or Fc-Fc), two amino acidsubstitutions (replacing anionic residues with lysines) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 101 mayoptionally be provided with the C-terminal lysine removed.

This ActRIIA-Fc fusion polypeptide is encoded by the following nucleicacid (SEQ ID NO: 102).

(SEQ ID NO: 102) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCGCTATACT TGGTAGATCA GAAACTCAGG 101AGTGTCTTTT CTTTAATGCT AATTGGGAAA AAGACAGAAC CAATCAAACT 151 GGTGTTGAACCGTGTTATGG TGACAAAGAT AAACGGCGGC ATTGTTTTGC 201 TACCTGGAAG AATATTTCTGGTTCCATTGA AATAGTGAAA CAAGGTTGTT 251 GGCTGGATGA TATCAACTGC TATGACAGGACTGATTGTGT AGAAAAAAAA 301 GACAGCCCTG AAGTATATTT CTGTTGCTGT GAGGGCAATATGTGTAATGA 351 AAAGTTTTCT TATTTTCCGG AGATGGAAGT CACACAGCCC ACTTCAAATC401 CAGTTACACC TAAGCCACCC ACCGGTGGTG GAACTCACAC ATGCCCACCG 451TGCCCAGCAC CTGAACTCCT GGGGGGACCG TCAGTCTTCC TCTTCCCCCC 501 AAAACCCAAGGACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG 551 TGGTGGTGGA CGTGAGCCACGAAGACCCTG AGGTCAAGTT CAACTGGTAC 601 GTGGACGGCG TGGAGGTGCA TAATGCCAAGACAAAGCCGC GGGAGGAGCA 651 GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTCCTGCACCAGG 701 ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC751 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA 801ACCACAGGTG TACACCCTGC CCCCATCCCG GAAGGAGATG ACCAAGAACC 851 AGGTCAGCCTGACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC 901 GTGGAGTGGG AGAGCAATGGGCAGCCGGAG AACAACTACA AGACCACGCC 951 TCCCGTGCTG AAGTCCGACG GCTCCTTCTTCCTCTATAGC AAGCTCACCG 1001 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATGCTCCGTGATG 1051 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC1101 GGGTAAA

The mature ActRIIA-Fc fusion polypeptide sequence is as follows (SEQ IDNO: 103) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 103) 1 ILGRSETQEC LFFNANWEKD RTNQTGVEPC YGDKDKRRHCFATWKNISGS 51 IEIVKQGCWL DDINCYDRTD CVEKKDSPEV YFCCCEGNMC NEKFSYFPEM 101EVTQPTSNPV TPKPPTGGGT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI 151 SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV 201 SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP 251 SRKEMTKNQV SLTCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLKSDGS 301 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSLSPGK

As described in Example 1, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 137) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ActRIIA-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing lysineswith anionic residues) can be introduced into the monomeric Fcpolypeptide. The amino acid sequence of SEQ ID NO: 137 may optionally beprovided without the C-terminal lysine. This complementary Fcpolypeptide is encoded by the nucleic acid of SEQ ID NO: 138, and themature monomeric Fc polypeptide (SEQ ID NO: 139) may optionally beprovided with the C-terminal lysine removed.

The ActRIIA-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 103 and SEQ ID NO: 139, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ActRIIA-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in theActRIIA-Fc and Fc polypeptide sequences of SEQ ID NOs: 401-402 and425-426, respectively.

The ActRIIA-Fc fusion polypeptide (SEQ ID NO: 401) uses the TPA leaderand is as follows:

(SEQ ID NO: 401) 1 MDAMKRGLCC VLLLCGAVFV SPGAAILGRS ETQECLFFNANWEKDRTNQT 51 GVEPCYGDKD KRRHCFATWK NISGSIEIVK QGCWLDDINC YDRTDCVEKK 101DSPEVYFCCC EGNMCNEKFS YFPEMEVTQP TSNPVTPKPP TGGGTHTCPP 151 CPAPELLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY 201 VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL 251 PAPIEKTISK AKGQPREPQV YTLPPCREEMTKNQVSLWCL VKGFYPSDIA 301 VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQQGNVFSCSVM 351 HEALHNHYTQ KSLSLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ActRIIA-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a tryptophan) can be introducedinto the Fc domain of the ActRIIA fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 401 mayoptionally be provided with the C-terminal lysine removed.

The mature ActRIIA-Fc fusion polypeptide (SEQ ID NO: 402) is as followsand may optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 402) 1 ILGRSETQEC LFFNANWEKD RTNQTGVEPC YGDKDKRRHCFATWKNISGS 51 IEIVKQGCWL DDINCYDRTD CVEKKDSPEV YFCCCEGNMC NEKFSYFPEM 101EVTQPTSNPV TPKPPTGGGT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI 151 SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV 201 SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP 251 CREEMTKNQV SLWCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS 301 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSLSPGK

As described in Example 1, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 425) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ActRIIA-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the monomeric Fc polypeptide as indicated. The amino acid sequenceof SEQ ID NO: 425 and the mature G1Fc polypeptide (SEQ ID NO: 426) mayoptionally be provided with the C-terminal lysine removed.

The ActRIIA-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 402 and SEQ ID NO: 426, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ActRIIA-Fc:Fc.

Purification of various ActRIIA-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm ActRIIA-Fc heterodimeric complex describedabove with that of an ActRIIA-Fc homodimeric complex. The single-armActRIIA-Fc and homodimeric ActRIIA-Fc were independently captured ontothe system using an anti-Fc antibody. Ligands were injected and allowedto flow over the captured receptor protein. Results are summarized inthe table below, in which ligand off-rates (k_(d)) typically associatedwith the most effective ligand traps are denoted in bold.

Ligand binding of single-arm ActRIIA-Fc compared to ActRIIA-Fc homodimerActRIIA-Fc homodimer Single-arm ActRIIA-Fc k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) Ligand (l/Ms) (l/s) (pM) (l/Ms) (l/s) (pM) Activin A 1.4 × 10⁷ 6.2× 10 ⁻⁴ 45 3.0 × 10⁷ 9.0 × 10 ⁻⁴ 30 Activin B 7.9 × 10⁶ 2.0 × 10 ⁻⁴ 252.9 × 10⁷ 1.4 × 10⁻³ 46 BMP5 4.0 × 10⁶ 4.5 × 10⁻³ 1100 4.8 × 10⁷ 5.8 ×10⁻² 1200 BMP10 2.9 × 10⁷ 2.5 × 10⁻³ 86 2.3 × 10⁷ 5.9 × 10⁻³ 250 GDF81.4 × 10⁷ 1.4 × 10⁻³ 99 4.7 × 10⁶ 5.0 × 10⁻³ 1100 GDF11 2.6 × 10⁷ 7.2 ×10 ⁻⁴ 28 4.9 × 10⁷ 1.1 × 10⁻² 220

These comparative binding data indicate that single-arm ActRIIA-Fc hasdifferent ligand selectivity than homodimeric ActRIIA-Fc (and alsodifferent than single-arm ActRIIB-Fc or homomeric ActRIIB-Fc—see Example1). Whereas ActRIIA-Fc homodimer exhibits preferential binding toactivin B combined with strong binding to activin A and GDF11,single-arm ActRIIA-Fc has a reversed preference for activin A overactivin B combined with greatly enhanced selectivity for activin A overGDF11 (weak binder). See FIG. 8. In addition, single-arm ActRIIA-Fclargely retains the intermediate binding to GDF8 and BMP10 observed withActRIIA-Fc homodimer.

These results indicate that single-arm ActRIIA-Fc heterodimer is anantagonist with substantially altered ligand selectivity compared toActRIIA-Fc homodimer. Accordingly, single-arm ActRIIA-Fc will be moreuseful than ActRIIA-Fc homodimer in certain applications where suchantagonism is advantageous. Examples include therapeutic applicationswhere it is desirable to antagonize activin A preferentially overactivin B while minimizing antagonism of GDF11.

Together the foregoing examples demonstrate that type I or type IIreceptor polypeptides, when placed in the context of a single-armheteromeric protein complex, form novel binding pockets that exhibitaltered selectivity relative to either type of homomeric proteincomplex, allowing the formation of novel protein agents for possible useas therapeutic agents.

Example 4. Generation and Characterization of a Single-Arm BMPRII-FcHeterodimer

Applicants constructed a soluble single-arm BMPRII-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman BMPRII was fused to a separate Fc domain with a linker positionedbetween the extracellular domain and this second Fc domain. Theindividual constructs are referred to as monomeric Fc polypeptide andBMPRII-Fc fusion polypeptide, respectively, and the sequences for eachare provided below. Applicants also envision additional single-armBMPRII-Fc heterodimeric complexes comprising the extracellular domain ofBMPRII isoform A (SEQ ID NO: 72).

Formation of a single-arm BMPRII-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIM-Fcheterodimer in Example 1. In a first approach, illustrated in theBMPRII-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 107-109and 137-139, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The BMPRII-Fc fusion polypeptide employs the TPA leader and is asfollows:

(SEQ ID NO: 107) 1 MDAMKRGLCC VLLLCGAVFV SPGASQNQER LCAFKDPYQQDLGIGESRIS 51 HENGTILCSK GSTCYGLWEK SKGDINLVKQ GCWSHIGDPQ ECHYEECVVT 101TTPPSIQNGT YRFCCCSTDL CNVNFTENFP PPDTTPLSPP HSFNRDETGG 151 GTHTCPPCPAPELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP 201 EVKFNWYVDG VEVHNAKTKPREEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 251 KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSRKEMTKN QVSLTCLVKG 301 FYPSDIAVEW ESNGQPENNY KTTPPVLKSD GSFFLYSKLTVDKSRWQQGN 351 VFSCSVMHEA LHNHYTQKSL SLSPGK

The leader and linker sequences are underlined. To promote formation ofthe BMPRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (BMPRII-Fc:BMPRII-Fc or Fc:Fc), two amino acidsubstitutions (replacing anionic residues with lysines) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 107 mayoptionally be provided with the C-terminal lysine removed.

This BMPRII-Fc fusion polypeptide is encoded by the following nucleicacid (SEQ ID NO: 108).

(SEQ ID NO: 108) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCTCGCAGAA TCAAGAACGC CTATGTGCGT 101TTAAAGATCC GTATCAGCAA GACCTTGGGA TAGGTGAGAG TAGAATCTCT 151 CATGAAAATGGGACAATATT ATGCTCGAAA GGTAGCACCT GCTATGGCCT 201 TTGGGAGAAA TCAAAAGGGGACATAAATCT TGTAAAACAA GGATGTTGGT 251 CTCACATTGG AGATCCCCAA GAGTGTCACTATGAAGAATG TGTAGTAACT 301 ACCACTCCTC CCTCAATTCA GAATGGAACA TACCGTTTCTGCTGTTGTAG 351 CACAGATTTA TGTAATGTCA ACTTTACTGA GAATTTTCCA CCTCCTGACA401 CAACACCACT CAGTCCACCT CATTCATTTA ACCGAGATGA GACCGGTGGT 451GGAACTCACA CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC 501 GTCAGTCTTCCTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC 551 GGACCCCTGA GGTCACATGCGTGGTGGTGG ACGTGAGCCA CGAAGACCCT 601 GAGGTCAAGT TCAACTGGTA CGTGGACGGCGTGGAGGTGC ATAATGCCAA 651 GACAAAGCCG CGGGAGGAGC AGTACAACAG CACGTACCGTGTGGTCAGCG 701 TCCTCACCGT CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC751 AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA 801AGCCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC 851 GGAAGGAGATGACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC 901 TTCTATCCCA GCGACATCGCCGTGGAGTGG GAGAGCAATG GGCAGCCGGA 951 GAACAACTAC AAGACCACGC CTCCCGTGCTGAAGTCCGAC GGCTCCTTCT 1001 TCCTCTATAG CAAGCTCACC GTGGACAAGA GCAGGTGGCAGCAGGGGAAC 1051 GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA1101 GAAGAGCCTC TCCCTGTCTC CGGGTAAA

The mature BMPRII-Fc fusion polypeptide sequence is as follows (SEQ IDNO: 109) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 109) 1 SQNQERLCAF KDPYQQDLGI GESRISHENG TILCSKGSTCYGLWEKSKGD 51 INLVKQGCWS HIGDPQECHY EECVVTTTPP SIQNGTYRFC CCSTDLCNVN 101FTENFPPPDT TPLSPPHSFN RDETGGGTHT CPPCPAPELL GGPSVFLFPP 151 KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 201 YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE 251 PQVYTLPPSR KEMTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP 301 PVLKSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP 351 GK

As described in Example 1, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 137) uses the TPA leader and incorporatesan optional N-terminal extension. To promote formation of theBMPRII-Fc:Fc heterodimer rather than either of the possible homodimericcomplexes, two amino acid substitutions (replacing lysines with anionicresidues) can be introduced into the monomeric Fc polypeptide. The aminoacid sequence of SEQ ID NO: 137 may optionally be provided with theC-terminal lysine removed. This complementary Fc polypeptide is encodedby the nucleic acid of SEQ ID NO: 138), and the mature monomeric Fcpolypeptide (SEQ ID NO: 139) may optionally be provided with theC-terminal lysine removed.

The BMPRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 103 and SEQ ID NO: 139, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising BMPRII-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the BMPRII-Fcand Fc polypeptide sequences of SEQ ID NOs: 405-406 and 425-426,respectively.

The BMPRII-Fc fusion polypeptide (SEQ ID NO: 405) uses the TPA leaderand is as follows:

(SEQ ID NO: 405) 1 MDAMKRGLCC VLLLCGAVFV SPGASQNQER LCAFKDPYQQDLGIGESRIS 51 HENGTILCSK GSTCYGLWEK SKGDINLVKQ GCWSHIGDPQ ECHYEECVVT 101TTPPSIQNGT YRFCCCSTDL CNVNFTENFP PPDTTPLSPP HSFNRDETGG 151 GTHTCPPCPAPELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP 201 EVKFNWYVDG VEVHNAKTKPREEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 251 KVSNKALPAP IEKTISKAKG QPREPQVYTLPPCREEMTKN QVSLWCLVKG 301 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLTVDKSRWQQGN 351 VFSCSVMHEA LHNHYTQKSL SLSPGK

The leader sequence and linker are underlined. To promote formation ofthe BMPRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a tryptophan) can be introducedinto the Fc domain of the BMPRII fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 405 mayoptionally be provided with the C-terminal lysine removed.

The mature BMPRII-Fc fusion polypeptide (SEQ ID NO: 406) is as followsand may optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 406) 1 SQNQERLCAF KDPYQQDLGI GESRISHENG TILCSKGSTCYGLWEKSKGD 51 INLVKQGCWS HIGDPQECHY EECVVTTTPP SIQNGTYRFC CCSTDLCNVN 101FTENFPPPDT TPLSPPHSFN RDETGGGTHT CPPCPAPELL GGPSVFLFPP 151 KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 201 YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE 251 PQVYTLPPCR EEMTKNQVSL WCLVKGFYPSDIAVEWESNG QPENNYKTTP 301 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP 351 GK

As described in Example 1, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 425) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the BMPRII-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,four amino acid substitutions can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 425 andthe mature Fc polypeptide (SEQ ID NO: 426) may optionally be providedwith the C-terminal lysine removed.

The BMPRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 406 and SEQ ID NO: 426, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising BMPRII-Fc:Fc.

Purification of various BMPRII-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm BMPRII-Fc heterodimeric complex describedabove with that of an BMPRII-Fc homodimeric complex. The single-armBMPRII-Fc and homodimeric BMPRII-Fc were independently captured onto thesystem using an anti-Fc antibody. Ligands were injected and allowed toflow over the captured receptor protein. Results are summarized in thetable below.

Ligand binding by single-arm BMPRII-Fc compared to BMPRII-Fc homodimerBMPRII-Fc homodimer Single-arm BMPRII-Fc k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) Ligand (1/Ms) (1/s) (pM) (1/Ms) (1/s) (pM) Activin B 2.0 × 10⁷ 7.5× 10⁻² 3800 Minimal binding BMP2 Transient * >2 × 10⁶ No binding BMP4 —No binding BMP5 — 4.1 × 10⁵ 1.5 × 10⁻² 36000 BMP6 Transient * >8900 Nobinding BMP7 Transient * >38000 No binding BMP9 1.2 × 10⁷ 2.6 × 10⁻²2100 Minimal binding BMP10 2.6 × 10⁷ 2.5 × 10⁻³ 98 2.1 × 10⁷ 9.1 × 10⁻³430 BMP15 9.9 × 10⁶ 2.8 × 10⁻³ 280 7.1 × 10⁷ 6.7 × 10⁻² 940 GDF6Transient * >88000 Minimal binding GDF7 — Transient * >190000 *Indeterminate due to transient nature of interaction — Not tested

These comparative binding data indicate that single-arm BMPRII-Fcheterodimer retains binding to only a subset of ligands bound byBMPRII-Fc homodimer. In particular, while the single-arm BMPRII-Fcheterodimer retains binding to BMP10 and BMP15, binding to BMP9 isessentially eliminated.

Example 5. Generation of a Single-Arm MISRII-Fc Heterodimer

Applicants envision construction of a soluble single-arm MISRII-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the extracellulardomain of human MISRII is fused to a separate Fc domain with a linkerpositioned between the extracellular domain and this second Fc domain.The individual constructs are referred to as monomeric Fc polypeptideand MISRII-Fc fusion polypeptide, respectively, and the sequences foreach are provided below. Applicants also envision additional single-armMISRII-Fc heterodimeric complexes comprising the extracellular domain ofMISRII isoform 2 or 3 (SEQ ID NOs: 76, 80).

Formation of a single-arm MISRII-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIM-Fcheterodimer in Example 1. In a first approach, illustrated in theMISRII-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 110-112and 137-139, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The MISRII-Fc fusion polypeptide employs the TPA leader and is asfollows:

(SEQ ID NO: 110) 1 MDAMKRGLCC VLLLCGAVFV SPGAPPNRRT CVFFEAPGVRGSTKTLGELL 51 DTGTELPRAI RCLYSRCCFG IWNLTQDRAQ VEMQGCRDSD EPGCESLHCD 101PSPRAHPSPG STLFTCSCGT DFCNANYSHL PPPGSPGTPG SQGPQAAPGE 151SIWMALTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV 201 VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD 251 WLNGKEYKCK VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSRKEMTKNQ 301 VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLKSDG SFFLYSKLTV 351 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

The leader and linker sequences are underlined. To promote formation ofthe MISRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (MISRII-Fc:MISRII-Fc or Fc:Fc), two amino acidsubstitutions (replacing anionic residues with lysines) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 110 mayoptionally be provided with the C-terminal lysine removed.

The mature MISRII-Fc fusion polypeptide sequence is as follows (SEQ IDNO: 112) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 112) 1 PPNRRTCVFF EAPGVRGSTK TLGELLDTGT ELPRAIRCLYSRCCFGIWNL 51 TQDRAQVEMQ GCRDSDEPGC ESLHCDPSPR AHPSPGSTLF TCSCGTDFCN 101ANYSHLPPPG SPGTPGSQGP QAAPGESIWM ALTGGGTHTC PPCPAPELLG 151 GPSVFLFPPKPKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN 201 AKTKPREEQY NSTYRVVSVLTVLHQDWLNG KEYKCKVSNK ALPAPIEKTI 251 SKAKGQPREP QVYTLPPSRK EMTKNQVSLTCLVKGFYPSD IAVEWESNGQ 301 PENNYKTTPP VLKSDGSFFL YSKLTVDKSR WQQGNVFSCSVMHEALHNHY 351 TQKSLSLSPG K

As described in Example 1, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 137) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe MISRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing lysineswith anionic residues) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 137 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 138, and the mature monomeric Fc polypeptide (SEQ ID NO: 139) mayoptionally be provided with the C-terminal lysine removed.

The MISRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 112 and SEQ ID NO: 139, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising MISRII-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the MISRII-Fcand Fc polypeptide sequences of SEQ ID NOs: 407-408 and 425-426,respectively.

The MISRII-Fc fusion polypeptide (SEQ ID NO: 407) uses the TPA leaderand is as follows:

(SEQ ID NO: 407) 1 MDAMKRGLCC VLLLCGAVFV SPGAPPNRRT CVFFEAPGVRGSTKTLGELL 51 DTGTELPRAI RCLYSRCCFG IWNLTQDRAQ VEMQGCRDSD EPGCESLHCD 101PSPRAHPSPG STLFTCSCGT DFCNANYSHL PPPGSPGTPG SQGPQAAPGE 151SIWMALTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV 201 VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD 251 WLNGKEYKCK VSNKALPAPIEKTISKAKGQ PREPQVYTLP PCREEMTKNQ 301 VSLWCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV 351 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

The leader sequence and linker are underlined. To promote formation ofthe MISRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a tryptophan) can be introducedinto the Fc domain of the MISRII fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 407 mayoptionally be provided with the C-terminal lysine removed.

The mature MISRII-Fc fusion polypeptide (SEQ ID NO: 408) is as followsand may optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 408) 1 PPNRRTCVFF EAPGVRGSTK TLGELLDTGT ELPRAIRCLYSRCCFGIWNL 51 TQDRAQVEMQ GCRDSDEPGC ESLHCDPSPR AHPSPGSTLF TCSCGTDFCN 101ANYSHLPPPG SPGTPGSQGP QAAPGESIWM ALTGGGTHTC PPCPAPELLG 151 GPSVFLFPPKPKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN 201 AKTKPREEQY NSTYRVVSVLTVLHQDWLNG KEYKCKVSNK ALPAPIEKTI 251 SKAKGQPREP QVYTLPPCRE EMTKNQVSLWCLVKGFYPSD IAVEWESNGQ 301 PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCSVMHEALHNHY 351 TQKSLSLSPG K

As described in Example 1, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 425) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the MISRII-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,four amino acid substitutions can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 425 andthe mature Fc polypeptide (SEQ ID NO: 426) may optionally be providedwith the C-terminal lysine removed.

The MISRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 408 and SEQ ID NO: 426, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising MISRII-Fc:Fc.

Purification of various MISRII-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 6. Generation and Characterization of a Single-Arm TGFβRII-FcHeterodimer

Applicants constructed a soluble single-arm TGFβRII-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman TGFβRII (short isoform, SEQ ID NO: 43) was fused to a separate Fcdomain with a linker positioned between the extracellular domain andthis second Fc domain. The individual constructs are referred to asmonomeric Fc polypeptide and TGFβRII-Fc fusion polypeptide,respectively, and the sequences for each are provided below. Applicantsalso envision additional single-arm TGFβRII-Fc complexes comprising theextracellular domain of TGFβRII isoform A (SEQ ID NO: 68) as well assingle-arm TGFβRII-Fc complexes in which the extracellular domain ofcanonical TGFβRII (short isoform, SEQ ID NO: 43) or that of TGFβRIIisoform A (SEQ ID NO: 68) contain a 36-amino-acid insert (SEQ ID NO: 95)derived from TGFβRII isoform C as described herein.

Formation of a single-arm TGFβRII-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theTGFβRII-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 113-115and 137-139, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The TGFβRII-Fc fusion polypeptide employs the TPA leader and is asfollows:

(SEQ ID NO: 113) 1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSVNNDMIVTDNNGAVKFP 51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151 FSEEYNTSNPDTGGGTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP 201 EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT 251 VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSRKE 301 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLKSDGSFFLY 351 SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

The leader and linker sequences are underlined. To promote formation ofthe TGFβRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (TGFβRII-Fc: TGFβRII-Fc or Fc:Fc), two amino acidsubstitutions (replacing anionic residues with lysines) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 113 mayoptionally be provided with the C-terminal lysine removed.

This TGFβRII-Fc fusion polypeptide is encoded by the following nucleicacid (SEQ ID NO: 114).

(SEQ ID NO: 114) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151 CAACTGTGTAAATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201 ATCCTGCATG AGCAACTGCAGCATCACCTC CATCTGTGAG AAGCCACAGG 251 AAGTCTGTGT GGCTGTATGG AGAAAGAATGACGAGAACAT AACACTAGAG 301 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTATTCTGGAAGA 351 TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA401 CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451TTCTCAGAAG AATATAACAC CAGCAATCCT GACACCGGTG GTGGAACTCA 501 CACATGCCCACCGTGCCCAG CACCTGAACT CCTGGGGGGA CCGTCAGTCT 551 TCCTCTTCCC CCCAAAACCCAAGGACACCC TCATGATCTC CCGGACCCCT 601 GAGGTCACAT GCGTGGTGGT GGACGTGAGCCACGAAGACC CTGAGGTCAA 651 GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCCAAGACAAAGC 701 CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC751 GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC 801CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG 851 GGCAGCCCCGAGAACCACAG GTGTACACCC TGCCCCCATC CCGGAAGGAG 901 ATGACCAAGA ACCAGGTCAGCCTGACCTGC CTGGTCAAAG GCTTCTATCC 951 CAGCGACATC GCCGTGGAGT GGGAGAGCAATGGGCAGCCG GAGAACAACT 1001 ACAAGACCAC GCCTCCCGTG CTGAAGTCCG ACGGCTCCTTCTTCCTCTAT 1051 AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC1101 ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC 1151TCTCCCTGTC TCCGGGTAAA

The mature TGFβRII-Fc fusion polypeptide sequence is as follows (SEQ IDNO: 115) and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 115) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCDNQKSCMSNCS 51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPDTGG GTHTCPPCPA 151 PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 201 VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 251 IEKTISKAKG QPREPQVYTL PPSRKEMTKNQVSLTCLVKG FYPSDIAVEW 301 ESNGQPENNY KTTPPVLKSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA 351 LHNHYTQKSL SLSPGK

As described in Example 1, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 137) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe TGFβRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing lysineswith anionic residues) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 137 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 138, and the mature monomeric Fc polypeptide (SEQ ID NO: 139) mayoptionally be provided with the C-terminal lysine removed.

The TGFβRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 115 and SEQ ID NO: 139, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising TGFβRII-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in theTGFβRII-Fc and Fc polypeptide sequences of SEQ ID NOs: 409-410 and425-426, respectively.

The TGFβRII-Fc fusion polypeptide (SEQ ID NO: 409) uses the TPA leaderand is as follows:

(SEQ ID NO: 409) 1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSVNNDMIVTDNNGAVKFP 51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151 FSEEYNTSNPDTGGGTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP 201 EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT 251 VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPCREE 301 MTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY 351 SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

The leader sequence and linker are underlined. To promote formation ofthe TGFβRII-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a tryptophan) can be introducedinto the Fc domain of the TGFβRII fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 409 mayoptionally be provided with the C-terminal lysine removed.

The mature TGFβRII-Fc fusion polypeptide (SEQ ID NO: 410) is as followsand may optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 410) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCDNQKSCMSNCS 51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPDTGG GTHTCPPCPA 151 PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 201 VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 251 IEKTISKAKG QPREPQVYTL PPCREEMTKNQVSLWCLVKG FYPSDIAVEW 301 ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA 351 LHNHYTQKSL SLSPGK

As described in Example 1, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 425) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the TGFβRII-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,four amino acid substitutions can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 425 andthe mature monomeric Fc polypeptide (SEQ ID NO: 426) may optionally beprovided with the C-terminal lysine removed.

The TGFβRII-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 410 and SEQ ID NO: 426, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising TGFβRII-Fc:Fc.

Purification of various TGFβRII-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm TGFβRII-Fc heterodimeric complex describedabove with that of an TGFβRII-Fc homodimeric complex. The single-armTGFβRII-Fc and homodimeric TGFβRII-Fc were independently captured ontothe system using an anti-Fc antibody. Ligands were injected and allowedto flow over the captured receptor protein. Results are summarized inthe table below.

Ligand binding by single-arm TGFβRII-Fc compared to TGFβRII-Fc homodimerTGFβRII-Fc homodimer Single-arm TGFβRII-Fc k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) Ligand (1/Ms) (1/s) (pM) (1/Ms) (1/s) (pM) TGFβ1 4.2 × 10⁷ 1.1 ×10⁻³ 25 1.5 × 10⁸ 4.7 × 10⁻³ 31 TGFβ2 Transient* >44000Transient* >61000 TGFβ3 5.9 × 10⁷ 5.9 × 10⁻³ 99 1.4 × 10⁸ 9.9 × 10⁻³ 73*Indeterminate due to transient nature of interaction

Example 7. Generation and Characterization of a Single-Arm ALK1-FcHeterodimer

Applicants constructed a soluble single-arm ALK1-Fc heterodimericcomplex comprising a monomeric Fc polypeptide with a short N-terminalextension and a second polypeptide in which the extracellular domain ofhuman ALK1 was fused to a separate Fc domain with a linker positionedbetween the extracellular domain and this second Fc domain. Theindividual constructs are referred to as monomeric Fc polypeptide andALK1-Fc fusion polypeptide, respectively, and the sequences for each areprovided below.

Formation of a single-arm ALK1-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIM-Fcheterodimer in Example 1. In a first approach, illustrated in theALK1-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 116-118and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK1-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 116) 1 MDAMKRGLCC VLLLCGAVFV SPGADPVKPS RGPLVTCTCESPHCKGPTCR 51 GAWCTVVLVR EEGRHPQEHR GCGNLHRELC RGRPTEFVNH YCCDSHLCNH 101NVSLVLEATQ PPSEQPGTDG QLATGGGTHT CPPCPAPELL GGPSVFLFPP 151 KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 201 YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE 251 PQVYTLPPSR EEMTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYDTTP 301 PVLDSDGSFF LYSDLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP 351 G

The leader and linker sequences are underlined. To promote formation ofthe ALK1-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK1-Fc:ALK1-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 116 mayoptionally be provided with a lysine added at the C-terminus.

This ALK1-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 117).

(SEQ ID NO: 117) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCGACCCTGT GAAGCCGTCT CGGGGCCCGC 101TGGTGACCTG CACGTGTGAG AGCCCACATT GCAAGGGGCC TACCTGCCGG 151 GGGGCCTGGTGCACAGTAGT GCTGGTGCGG GAGGAGGGGA GGCACCCCCA 201 GGAACATCGG GGCTGCGGGAACTTGCACAG GGAGCTCTGC AGGGGCCGCC 251 CCACCGAGTT CGTCAACCAC TACTGCTGCGACAGCCACCT CTGCAACCAC 301 AACGTGTCCC TGGTGCTGGA GGCCACCCAA CCTCCTTCGGAGCAGCCGGG 351 AACAGATGGC CAGCTGGCCA CCGGTGGTGG AACTCACACA TGCCCACCGT401 GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA 451AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT 501 GGTGGTGGACGTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG 551 TGGACGGCGT GGAGGTGCATAATGCCAAGA CAAAGCCGCG GGAGGAGCAG 601 TACAACAGCA CGTACCGTGT GGTCAGCGTCCTCACCGTCC TGCACCAGGA 651 CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAACAAAGCCCTCC 701 CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA751 CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA 801GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG 851 TGGAGTGGGAGAGCAATGGG CAGCCGGAGA ACAACTACGA CACCACGCCT 901 CCCGTGCTGG ACTCCGACGGCTCCTTCTTC CTCTATAGCG ACCTCACCGT 951 GGACAAGAGC AGGTGGCAGC AGGGGAACGTCTTCTCATGC TCCGTGATGC 1001 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTCCCTGTCTCCG 1051 GGT

The mature ALK1-Fc fusion polypeptide sequence is as follows (SEQ ID NO:118) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 118) 1 DPVKPSRGPL VTCTCESPHC KGPTCRGAWC TVVLVREEGRHPQEHRGCGN 51 LHRELCRGRP TEFVNHYCCD SHLCNHNVSL VLEATQPPSE QPGTDGQLAT 101GGGTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 151 DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 201 KCKVSNKALP APIEKTISKAKGQPREPQVY TLPPSREEMT KNQVSLTCLV 251 KGFYPSDIAV EWESNGQPEN NYDTTPPVLDSDGSFFLYSD LTVDKSRWQQ 301 GNVFSCSVMH EALHNHYTQK SLSLSPG

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK1-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc polypeptide (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK1-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 118 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK1-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion proteins, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK1-Fcand Fc polypeptide sequences of SEQ ID NOs: 411-412 and 427-428,respectively.

The ALK1-Fc fusion polypeptide (SEQ ID NO: 411) uses the TPA leader andis as follows:

(SEQ ID NO: 411) 1 MDAMKRGLCC VLLLCGAVFV SPGADPVKPS RGPLVTCTCESPHCKGPTCR 51 GAWCTVVLVR EEGRHPQEHR GCGNLHRELC RGRPTEFVNH YCCDSHLCNH 101NVSLVLEATQ PPSEQPGTDG QLATGGGTHT CPPCPAPELL GGPSVFLFPP 151 KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 201 YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE 251 PQVCTLPPSR EEMTKNQVSL SCAVKGFYPSDIAVEWESNG QPENNYKTTP 301 PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP 351 GK

The leader sequence and linker are underlined. To promote formation ofthe ALK1-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK1 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 411 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK1-Fc fusion polypeptide (SEQ ID NO: 412) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 412) 1 DPVKPSRGPL VTCTCESPHC KGPTCRGAWC TVVLVREEGRHPQEHRGCGN 51 LHRELCRGRP TEFVNHYCCD SHLCNHNVSL VLEATQPPSE QPGTDGQLAT 101GGGTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 151 DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 201 KCKVSNKALP APIEKTISKAKGQPREPQVC TLPPSREEMT KNQVSLSCAV 251 KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLVSK LTVDKSRWQQ 301 GNVFSCSVMH EALHNHYTQK SLSLSPGK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK1-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature Fc polypeptide (SEQ ID NO: 428) may optionally be providedwith the C-terminal lysine removed.

The ALK1-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 412 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK1-Fc:Fc.

Purification of various ALK1-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the single-arm ALK1-Fc heterodimeric complex describedabove with that of an ALK1-Fc homodimeric complex. The single-armALK1-Fc and homodimeric ALK1-Fc were independently captured onto thesystem using an anti-Fc antibody. Ligands were injected and allowed toflow over the captured receptor protein. Results are summarized in thetable below, in which ligand off-rates (k_(d)) typically associated withthe most effective ligand traps are denoted in bold.

Ligand binding by single-arm ALK1-Fc compared to ALK1-Fc homodimerALK1-Fc homodimer Single-arm ALK1-Fc k_(a) k_(d) K_(D) k_(a) k_(d) K_(D)Ligand (l/Ms) (l/s) (pM) (l/Ms) (1/s) (pM) BMP9 7.9 × 10⁶ 1.3 × 10 ⁻⁴ 161.2 × 10⁷ 1.7 × 10⁻³ 140 BMP10 1.7 × 10⁷ 1.1 × 10 ⁻⁴ 6 2.8 × 10⁶ 3.0 ×10 ⁻⁴ 100

These comparative binding data indicate that single-arm ALK1-Fc hasaltered ligand selectivity compared to homodimeric ALK1-Fc. Single-armALK1-FRc retains the strong binding to BMP10 observed with homodimericALK1-Fc while binding BMP9 less tightly than does ALK1-Fc homodimer, asthe off-rate of BMP9 binding to single-arm ALK1-Fc is approximately10-fold faster than it is for binding to homodimeric AK1-Fc. Theseresults indicate that single-arm ALK1-Fc is a more selective antagonistthan ActRIIB-Fc homodimer. Accordingly, single-arm ALK1-Fc will be moreuseful than homodimeric ALK1-Fc in certain applications where suchselective antagonism is advantageous. Examples include therapeuticapplications where it is desirable to retain antagonism of BMP10 butreduce antagonism of BMP9.

Example 8. Generation of a Single-Arm ALK2-Fc Heterodimer

Applicants envision construction of a soluble single-arm ALK2-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the extracellulardomain of human ALK2 is fused to a separate Fc domain with a linkerpositioned between the extracellular domain and this second Fc domain.The individual constructs are referred to as monomeric Fc polypeptideand ALK2-Fc fusion polypeptide, respectively, and the sequences for eachare provided below.

Formation of a single-arm ALK2-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIM-Fcheterodimer in Example 1. In a first approach, illustrated in theALK2-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 119-121and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK2-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 119) 1 MDAMKRGLCC VLLLCGAVFV SPGAMEDEKP KVNPKLYMCVCEGLSCGNED 51 HCEGQQCFSS LSINDGFHVY QKGCFQVYEQ GKMTCKTPPS PGQAVECCQG 101DWCNRNITAQ LPTKGKSFPG TQNFHLETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG 251 QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNY 301 DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEALHNHYTQKSL 351 SLSPG

The leader and linker sequences are underlined. To promote formation ofthe ALK2-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK2-Fc:ALK2-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 119 mayoptionally be provided with a lysine added at the C-terminus.

This ALK2-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 120).

(SEQ ID NO: 120) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCATGGAAGA TGAGAAGCCC AAGGTCAACC 101CCAAACTCTA CATGTGTGTG TGTGAAGGTC TCTCCTGCGG TAATGAGGAC 151 CACTGTGAAGGCCAGCAGTG CTTTTCCTCA CTGAGCATCA ACGATGGCTT 201 CCACGTCTAC CAGAAAGGCTGCTTCCAGGT TTATGAGCAG GGAAAGATGA 251 CCTGTAAGAC CCCGCCGTCC CCTGGCCAAGCTGTGGAGTG CTGCCAAGGG 301 GACTGGTGTA ACAGGAACAT CACGGCCCAG CTGCCCACTAAAGGAAAATC 351 CTTCCCTGGA ACACAGAATT TCCACTTGGA GACCGGTGGT GGAACTCACA401 CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501 GGTCACATGCGTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551 TCAACTGGTA CGTGGACGGCGTGGAGGTGC ATAATGCCAA GACAAAGCCG 601 CGGGAGGAGC AGTACAACAG CACGTACCGTGTGGTCAGCG TCCTCACCGT 651 CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGCAAGGTCTCCA 701 ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG751 CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT 801GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA 851 GCGACATCGCCGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC 901 GACACCACGC CTCCCGTGCTGGACTCCGAC GGCTCCTTCT TCCTCTATAG 951 CGACCTCACC GTGGACAAGA GCAGGTGGCAGCAGGGGAAC GTCTTCTCAT 1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCAGAAGAGCCTC 1051 TCCCTGTCTC CGGGT

The mature ALK2-Fc fusion polypeptide sequence is as follows (SEQ ID NO:121) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 121) 1 MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSINDGFHVYQKGC 51 FQVYEQGKMT CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF 101HLETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151 VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201 GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR EEMTKNQVSL 251 TCLVKGFYPS DIAVEWESNG QPENNYDTTPPVLDSDGSFF LYSDLTVDKS 301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK2-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc polypeptide (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK2-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 121 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK2-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK2-Fcand Fc polypeptide sequences of SEQ ID NOs: 413-414 and 427-428,respectively.

The ALK2-Fc fusion polypeptide (SEQ ID NO: 413) uses the TPA leader andis as follows:

(SEQ ID NO: 413) 1 MDAMKRGLCC VLLLCGAVFV SPGAMEDEKP KVNPKLYMCVCEGLSCGNED 51 HCEGQQCFSS LSINDGFHVY QKGCFQVYEQ GKMTCKTPPS PGQAVECCQG 101DWCNRNITAQ LPTKGKSFPG TQNFHLETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG 251 QPREPQVCTL PPSREEMTKN QVSLSCAVKGFYPSDIAVEW ESNGQPENNY 301 KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEALHNHYTQKSL 351 SLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ALK2-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK2 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 413 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK2-Fc fusion polypeptide (SEQ ID NO: 414) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 414) 1 MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSINDGFHVYQKGC 51 FQVYEQGKMT CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF 101HLETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151 VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201 GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVCTLPPSR EEMTKNQVSL 251 SCAVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LVSKLTVDKS 301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK2-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature Fc polypeptide (SEQ ID NO: 428) may optionally be providedwith the C-terminal lysine removed.

The ALK2-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 414 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK2-Fc:Fc.

Purification of various ALK2-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 9. Generation of a Single-Arm ALK4-Fc Heterodimer

Applicants envision construction of a soluble single-arm ALK4-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the extracellulardomain of human ALK4 is fused to a separate Fc domain with a linkerpositioned between the extracellular domain and this second Fc domain.The individual constructs are referred to as monomeric Fc polypeptideand ALK4-Fc fusion polypeptide, respectively, and the sequences for eachare provided below. Applicants also envision additional single-armALK4-Fc heterodimeric complexes comprising the extracellular domain ofALK4 isoform B (SEQ ID NO: 84).

Formation of a single-arm ALK4-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theALK4-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 125-127and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK4-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 125) 1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSCLQANYTCETD 51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD 101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG 251 QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNY 301 DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEALHNHYTQKSL 351 SLSPG

The leader and linker sequences are underlined. To promote formation ofthe ALK4-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK4-Fc:ALK4-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 125 mayoptionally be provided with a lysine added at the C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 126).

(SEQ ID NO: 126) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC 101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT 151 GGGGCCTGCATGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT 201 GCGCACCTGC ATCCCCAAAGTGGAGCTGGT CCCTGCCGGG AAGCCCTTCT 251 ACTGCCTGAG CTCGGAGGAC CTGCGCAACACCCACTGCTG CTACACTGAC 301 TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACCTCAAGGAGCC 351 TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA401 CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501 GGTCACATGCGTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551 TCAACTGGTA CGTGGACGGCGTGGAGGTGC ATAATGCCAA GACAAAGCCG 601 CGGGAGGAGC AGTACAACAG CACGTACCGTGTGGTCAGCG TCCTCACCGT 651 CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGCAAGGTCTCCA 701 ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG751 CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT 801GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA 851 GCGACATCGCCGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC 901 GACACCACGC CTCCCGTGCTGGACTCCGAC GGCTCCTTCT TCCTCTATAG 951 CGACCTCACC GTGGACAAGA GCAGGTGGCAGCAGGGGAAC GTCTTCTCAT 1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCAGAAGAGCCTC 1051 TCCCTGTCTC CGGGT

The mature ALK4-Fc fusion polypeptide sequence is as follows (SEQ ID NO:127) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 127) 1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGMEHHVRTCIPKV 51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG 101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151 VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201 GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR EEMTKNQVSL 251 TCLVKGFYPS DIAVEWESNG QPENNYDTTPPVLDSDGSFF LYSDLTVDKS 301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK4-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc polypeptide (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK4-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 127 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK4-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK4-Fcand Fc polypeptide sequences of SEQ ID NOs: 417-418 and 427-428,respectively.

The ALK4-Fc fusion polypeptide (SEQ ID NO: 417) uses the TPA leader andis as follows:

(SEQ ID NO: 417) 1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSCLQANYTCETD 51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD 101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG 251 QPREPQVCTL PPSREEMTKN QVSLSCAVKGFYPSDIAVEW ESNGQPENNY 301 KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEALHNHYTQKSL 351 SLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ALK4-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK4 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 417 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK4-Fc fusion polypeptide (SEQ ID NO: 418) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 418) 1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGMEHHVRTCIPKV 51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG 101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151 VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201 GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVCTLPPSR EEMTKNQVSL 251 SCAVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LVSKLTVDKS 301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK4-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature Fc polypeptide (SEQ ID NO: 428) may optionally be providedwith the C-terminal lysine removed.

The ALK4-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 418 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK4-Fc:Fc.

Purification of various ALK4-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 10. Generation of a Single-Arm ALK5-Fc Heterodimer

Applicants envision construction of a soluble single-arm ALK5-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the extracellulardomain of human ALK5 is fused to a separate Fc domain with a linkerpositioned between the extracellular domain and this second Fc domain.The individual constructs are referred to as monomeric Fc polypeptideand ALK5-Fc fusion polypeptide, respectively, and the sequences for eachare provided below. Applicants also envision additional single-armALK5-Fc heterodimeric complexes comprising the extracellular domain ofALK5 isoform 2 (SEQ ID NO: 88).

Formation of a single-arm ALK5-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theALK5-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 128-130and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK5-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 128) 1 MDAMKRGLCC VLLLCGAVFV SPGAALLPGA TALQCFCHLCTKDNFTCVTD 51 GLCFVSVTET TDKVIHNSMC IAEIDLIPRD RPFVCAPSSK TGSVTTTYCC 101NQDHCNKIEL PTTVKSSPGL GPVETGGGTH TCPPCPAPEL LGGPSVFLFP 151 PKPKDTLMISRTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE 201 QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR 251 EPQVYTLPPS REEMTKNQVS LTCLVKGFYPSDIAVEWESN GQPENNY D TT 301 PPVLDSDGSF FLYS D LTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLS 351 PG

The leader and linker sequences are underlined. To promote formation ofthe ALK5-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK5-Fc:ALK5-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated.The amino acid sequence of SEQ ID NO: 128 may optionally be providedwith a lysine added at the C-terminus.

This ALK5-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 129).

(SEQ ID NO: 129) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCGCGCTGCT CCCGGGGGCG ACGGCGTTAC 101AGTGTTTCTG CCACCTCTGT ACAAAAGACA ATTTTACTTG TGTGACAGAT 151 GGGCTCTGCTTTGTCTCTGT CACAGAGACC ACAGACAAAG TTATACACAA 201 CAGCATGTGT ATAGCTGAAATTGACTTAAT TCCTCGAGAT AGGCCGTTTG 251 TATGTGCACC CTCTTCAAAA ACTGGGTCTGTGACTACAAC ATATTGCTGC 301 AATCAGGACC ATTGCAATAA AATAGAACTT CCAACTACTGTAAAGTCATC 351 ACCTGGCCTT GGTCCTGTGG AAACCGGTGG TGGAACTCAC ACATGCCCAC401 CGTGCCCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC 451CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG 501 CGTGGTGGTGGACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT 551 ACGTGGACGG CGTGGAGGTGCATAATGCCA AGACAAAGCC GCGGGAGGAG 601 CAGTACAACA GCACGTACCG TGTGGTCAGCGTCCTCACCG TCCTGCACCA 651 GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCCAACAAAGCCC 701 TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG GCAGCCCCGA751 GAACCACAGG TGTACACCCT GCCCCCATCC CGGGAGGAGA TGACCAAGAA 801CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG 851 CCGTGGAGTGGGAGAGCAAT GGGCAGCCGG AGAACAACTA CGACACCACG 901 CCTCCCGTGC TGGACTCCGACGGCTCCTTC TTCCTCTATA GCGACCTCAC 951 CGTGGACAAG AGCAGGTGGC AGCAGGGGAACGTCTTCTCA TGCTCCGTGA 1001 TGCATGAGGC TCTGCACAAC CACTACACGC AGAAGAGCCTCTCCCTGTCT 1051 CCGGGT

The mature ALK5-Fc fusion polypeptide sequence is as follows (SEQ ID NO:130) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 130) 1 ALLPGATALQ CFCHLCTKDN FTCVTDGLCF VSVTETTDKVIHNSMCIAEI 51 DLIPRDRPFV CAPSSKTGSV TTTYCCNQDH CNKIELPTTV KSSPGLGPVE 101TGGGTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH 151 EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE 201 YKCKVSNKAL PAPIEKTISKAKGQPREPQV YTLPPSREEM TKNQVSLTCL 251 VKGFYPSDIA VEWESNGQPE NNYDTTPPVLDSDGSFFLYS DLTVDKSRWQ 301 QGNVFSCSVM HEALHNHYTQ KSLSLSPG

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK5-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc polypeptide (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK5-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 130 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK5-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK5-Fcand Fc polypeptide sequences of SEQ ID NOs: 419-420 and 427-428,respectively.

The ALK5-Fc fusion polypeptide (SEQ ID NO: 419) uses the TPA leader andis as follows:

(SEQ ID NO: 419) 1 MDAMKRGLCC VLLLCGAVFV SPGAALLPGA TALQCFCHLCTKDNFTCVTD 51 GLCFVSVTET TDKVIHNSMC IAEIDLIPRD RPFVCAPSSK TGSVTTTYCC 101NQDHCNKIEL PTTVKSSPGL GPVETGGGTH TCPPCPAPEL LGGPSVFLFP 151 PKPKDTLMISRTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE 201 QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR 251 EPQVCTLPPS REEMTKNQVS LSCAVKGFYPSDIAVEWESN GQPENNYKTT 301 PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHNHYTQKSLSLS 351 PGK

The leader sequence and linker are underlined. To promote formation ofthe ALK5-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK5 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 419 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK5-Fc fusion polypeptide (SEQ ID NO: 420) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 420) 1 ALLPGATALQ CFCHLCTKDN FTCVTDGLCF VSVTETTDKVIHNSMCIAEI 51 DLIPRDRPFV CAPSSKTGSV TTTYCCNQDH CNKIELPTTV KSSPGLGPVE 101TGGGTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH 151 EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE 201 YKCKVSNKAL PAPIEKTISKAKGQPREPQV CTLPPSREEM TKNQVSLSCA 251 VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLVS KLTVDKSRWQ 301 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK5-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature monomeric G1Fc polypeptide (SEQ ID NO: 428) may optionally beprovided with the C-terminal lysine removed.

The ALK5-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 420 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK5-Fc:Fc.

Purification of various ALK5-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 11. Generation of a Single-Arm ALK6-Fc Heterodimer

Applicants envision construction of a soluble single-arm ALK6-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the extracellulardomain of human ALK6 is fused to a separate Fc domain with a linkerpositioned between the extracellular domain and this second Fc domain.The individual constructs are referred to as monomeric Fc polypeptideand ALK6-Fc fusion polypeptide, respectively, and the sequences for eachare provided below. Applicants also envision additional single-armALK6-Fc heterodimeric complexes comprising the extracellular domain ofALK6 isoform 2 (SEQ ID NO: 92).

Formation of a single-arm ALK6-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theALK6-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 131-133and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK6-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 131) 1 MDAMKRGLCC VLLLCGAVFV SPGAKKEDGE STAPTPRPKVLRCKCHHHCP 51 EDSVNNICST DGYCFTMIEE DDSGLPVVTS GCLGLEGSDF QCRDTPIPHQ 101RRSIECCTER NECNKDLHPT LPPLKNRDFV DGPIHHRTGG GTHTCPPCPA 151 PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 201 VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 251 IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW 301 ESNGQPENNY D TTPPVLDSD GSFFLYS D LT VDKSRWQQGNVFSCSVMHEA 351 LHNHYTQKSL SLSPG

The leader and linker sequences are underlined. To promote formation ofthe ALK6-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK6-Fc:ALK6-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 131 mayoptionally be provided with a lysine added at the C-terminus.

This ALK6-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 132).

(SEQ ID NO: 132) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCAAGAAAGA GGATGGTGAG AGTACAGCCC 101CCACCCCCCG TCCAAAGGTC TTGCGTTGTA AATGCCACCA CCATTGTCCA 151 GAAGACTCAGTCAACAATAT TTGCAGCACA GACGGATATT GTTTCACGAT 201 GATAGAAGAG GATGACTCTGGGTTGCCTGT GGTCACTTCT GGTTGCCTAG 251 GACTAGAAGG CTCAGATTTT CAGTGTCGGGACACTCCCAT TCCTCATCAA 301 AGAAGATCAA TTGAATGCTG CACAGAAAGG AACGAATGTAATAAAGACCT 351 ACACCCTACA CTGCCTCCAT TGAAAAACAG AGATTTTGTT GATGGACCTA401 TACACCACAG GACCGGTGGT GGAACTCACA CATGCCCACC GTGCCCAGCA 451CCTGAACTCC TGGGGGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA 501 GGACACCCTCATGATCTCCC GGACCCCTGA GGTCACATGC GTGGTGGTGG 551 ACGTGAGCCA CGAAGACCCTGAGGTCAAGT TCAACTGGTA CGTGGACGGC 601 GTGGAGGTGC ATAATGCCAA GACAAAGCCGCGGGAGGAGC AGTACAACAG 651 CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAGGACTGGCTGA 701 ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC751 ATCGAGAAAA CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT 801GTACACCCTG CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC 851 TGACCTGCCTGGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG 901 GAGAGCAATG GGCAGCCGGAGAACAACTAC GACACCACGC CTCCCGTGCT 951 GGACTCCGAC GGCTCCTTCT TCCTCTATAGCGACCTCACC GTGGACAAGA 1001 GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGATGCATGAGGCT 1051 CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGT

The mature ALK6-Fc fusion polypeptide sequence is as follows (SEQ ID NO:133) and may optionally be provided with a lysine added at theC-terminus.

(SEQ ID NO: 133) 1 KKEDGESTAP TPRPKVLRCK CHHHCPEDSV NNICSTDGYCFTMIEEDDSG 51 LPVVTSGCLG LEGSDFQCRD TPIPHQRRSI ECCTERNECN KDLHPTLPPL 101KNRDFVDGPI HHRTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 151 TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 201 LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR 251 EEMTKNQVSL TCLVKGFYPS DIAVEWESNGQPENNYDTTP PVLDSDGSFF 301 LYSDLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK6-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc protein (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK6-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 133 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK6-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK6-Fcand Fc polypeptide sequences of SEQ ID NOs: 421-422 and 427-428,respectively.

The ALK6-Fc fusion polypeptide (SEQ ID NO: 421) uses the TPA leader andis as follows:

(SEQ ID NO: 421) 1 MDAMKRGLCC VLLLCGAVFV SPGAKKEDGE STAPTPRPKVLRCKCHHHCP 51 EDSVNNICST DGYCFTMIEE DDSGLPVVTS GCLGLEGSDF QCRDTPIPHQ 101RRSIECCTER NECNKDLHPT LPPLKNRDFV DGPIHHRTGG GTHTCPPCPA 151 PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 201 VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 251 IEKTISKAKG QPREPQVCTL PPSREEMTKNQVSLSCAVKG FYPSDIAVEW 301 ESNGQPENNY KTTPPVLDSD GSFFLVSKLT VDKSRWQQGNVFSCSVMHEA 351 LHNHYTQKSL SLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ALK6-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK6 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 421 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK6-Fc fusion polypeptide (SEQ ID NO: 422) is as follows andmay optionally be provided with the C-terminal lysine removed.

(SEQ ID NO: 422) 1 KKEDGESTAP TPRPKVLRCK CHHHCPEDSV NNICSTDGYCFTMIEEDDSG 51 LPVVTSGCLG LEGSDFQCRD TPIPHQRRSI ECCTERNECN KDLHPTLPPL 101KNRDFVDGPI HHRTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 151 TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 201 LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVCTLPPSR 251 EEMTKNQVSL SCAVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF 301 LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK6-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature monomeric G1Fc polypeptide (SEQ ID NO: 428) may optionally beprovided with the C-terminal lysine removed.

The ALK6-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 422 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK6-Fc:Fc.

Purification of various ALK6-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 12. Generation of a Single-Arm ALK7-Fc Heterodimer

Applicants envision construction of a soluble single-arm ALK7-Fcheterodimeric complex comprising a monomeric Fc polypeptide with a shortN-terminal extension and a second polypeptide in which the N-terminallytruncated (NM) extracellular domain of human ALK7 is fused to a separateFc domain with a linker positioned between the extracellular domain andthis second Fc domain. The individual constructs are referred to asmonomeric Fc polypeptide and ALK7-Fc fusion polypeptide, respectively,and the sequences for each are provided below. Applicants also envisionadditional single-arm ALK7-Fc heterodimeric complexes comprising otherN-terminally truncated variants (e.g., NA5 variant) of ALK7 isoform 1(SEQ ID NO: 313), the extracellular domain of ALK7 isoform 2 (SEQ ID NO:302), or native processed sequences of ALK7 isoforms 3 and 4 (SEQ IDNOs: 306, 310).

Formation of a single-arm ALK7-Fc heterodimer may be guided byapproaches similar to those described for single-arm ActRIIB-Fcheterodimer in Example 1. In a first approach, illustrated in theALK7-Fc and monomeric Fc polypeptide sequences of SEQ ID NOs: 134-136and 140-142, respectively, one Fc domain is altered to introducecationic amino acids at the interaction face, while the other Fc domainis altered to introduce anionic amino acids at the interaction face.

The ALK7-Fc fusion polypeptide employs the TPA leader and is as follows:

(SEQ ID NO: 134) 1 MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTCQTEGACWASV 51 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP 101TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 151 TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 201 LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR 251 EEMTKNQVSL TCLVKGFYPS DIAVEWESNGQPENNYDTTP PVLDSDGSFF 301 LYSDLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The leader and linker sequences are underlined. To promote formation ofthe ALK7-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes (ALK7-Fc:ALK7-Fc or Fc:Fc), two amino acidsubstitutions (replacing lysines with anionic amino acids) can beintroduced into the Fc domain of the fusion polypeptide as indicated bydouble underline above. The amino acid sequence of SEQ ID NO: 134 mayoptionally be provided with a lysine added at the C-terminus.

This ALK7-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 135).

(SEQ ID NO: 135) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC 51 AGTCTTCGTT TCGCCCGGCG CCGGACTGAA GTGTGTATGT CTTTTGTGTG 101ATTCTTCAAA CTTTACCTGC CAAACAGAAG GAGCATGTTG GGCATCAGTC 151 ATGCTAACCAATGGAAAAGA GCAGGTGATC AAATCCTGTG TCTCCCTTCC 201 AGAACTGAAT GCTCAAGTCTTCTGTCATAG TTCCAACAAT GTTACCAAAA 251 CCGAATGCTG CTTCACAGAT TTTTGCAACAACATAACACT GCACCTTCCA 301 ACAGCATCAC CAAATGCCCC AAAACTTGGA CCCATGGAGACCGGTGGTGG 351 AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT401 CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG 451ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA 501 GGTCAAGTTCAACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA 551 CAAAGCCGCG GGAGGAGCAGTACAACAGCA CGTACCGTGT GGTCAGCGTC 601 CTCACCGTCC TGCACCAGGA CTGGCTGAATGGCAAGGAGT ACAAGTGCAA 651 GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACCATCTCCAAAG 701 CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG751 GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT 801CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA 851 ACAACTACGACACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC 901 CTCTATAGCG ACCTCACCGTGGACAAGAGC AGGTGGCAGC AGGGGAACGT 951 CTTCTCATGC TCCGTGATGC ATGAGGCTCTGCACAACCAC TACACGCAGA 1001 AGAGCCTCTC CCTGTCTCCG GGT

The mature ALK7-Fc fusion polypeptide sequence is expected to be asfollows (SEQ ID NO: 136) and may optionally be provided with a lysineadded at the C-terminus.

(SEQ ID NO: 136) 1 GLKCVCLLCD SSNFTCQTEG ACWASVMLTN GKEQVIKSCVSLPELNAQVF 51 CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC 101PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 151 DGVEVHNAKTKPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP 201 APIEKTISKA KGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAV 251 EWESNGQPEN NYDTTPPVLD SDGSFFLYSDLTVDKSRWQQ GNVFSCSVMH 301 EALHNHYTQK SLSLSPG

As described in Example 2, the complementary form of monomeric humanG1Fc polypeptide (SEQ ID NO: 140) employs the TPA leader andincorporates an optional N-terminal extension. To promote formation ofthe ALK7-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing anionicresidues with lysines) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 140 mayoptionally be provided with the C-terminal lysine removed. Thiscomplementary Fc polypeptide is encoded by the nucleic acid of SEQ IDNO: 141, and the mature monomeric Fc polypeptide (SEQ ID NO: 142) mayoptionally be provided with the C-terminal lysine removed.

The ALK7-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 136 and SEQ ID NO: 142, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK7-Fc:Fc.

In another approach to promoting the formation of heteromultimercomplexes using asymmetric Fc fusion polypeptides, the Fc domains arealtered to introduce complementary hydrophobic interactions and anadditional intermolecular disulfide bond as illustrated in the ALK7-Fcand Fc polypeptide sequences of SEQ ID NOs: 423-424 and 427-428,respectively.

The ALK7-Fc fusion polypeptide (SEQ ID NO: 423) uses the TPA leader andis as follows:

(SEQ ID NO: 423) 1 MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTCQTEGACWASV 51 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP 101TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 151 TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 201 LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVCTLPPSR 251 EEMTKNQVSL SCAVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF 301 LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

The leader sequence and linker are underlined. To promote formation ofthe ALK7-Fc:Fc heterodimer rather than either of the possiblehomodimeric complexes, four amino acid substitutions can be introducedinto the Fc domain of the ALK7 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 423 mayoptionally be provided with the C-terminal lysine removed.

The mature ALK7-Fc fusion polypeptide (SEQ ID NO: 424) is expected to beas follows and may optionally be provided with the C-terminal lysineremoved.

(SEQ ID NO: 424) 1 GLKCVCLLCD SSNFTCQTEG ACWASVMLTN GKEQVIKSCVSLPELNAQVF 51 CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC 101PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 151 DGVEVHNAKTKPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP 201 APIEKTISKA KGQPREPQVCTLPPSREEMT KNQVSLSCAV KGFYPSDIAV 251 EWESNGQPEN NYKTTPPVLD SDGSFFLVSKLTVDKSRWQQ GNVFSCSVMH 301 EALHNHYTQK SLSLSPGK

As described in Example 2, the complementary form of monomeric G1Fcpolypeptide (SEQ ID NO: 427) employs the TPA leader and incorporates anoptional N-terminal extension. To promote formation of the ALK7-Fc:Fcheterodimer rather than either of the possible homodimeric complexes,two amino acid substitutions (replacing a serine with a cysteine and athreonine with a tryptophan) can be introduced into the monomeric Fcpolypeptide as indicated. The amino acid sequence of SEQ ID NO: 427 andthe mature monomeric G1Fc polypeptide (SEQ ID NO: 428) may optionally beprovided with the C-terminal lysine removed.

The ALK7-Fc fusion polypeptide and monomeric Fc polypeptide of SEQ IDNO: 424 and SEQ ID NO: 428, respectively, may be co-expressed andpurified from a CHO cell line to give rise to a single-arm heteromericprotein complex comprising ALK7-Fc:Fc.

Purification of various ALK7-Fc:Fc complexes could be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Together these examples demonstrate that type I or type II receptorpolypeptides, when placed in the context of a single-arm heteromericprotein complex, form novel binding pockets that exhibit alteredselectivity relative to a homodimeric complex of the same receptorpolypeptide, allowing the formation of novel protein agents for possibleuse as therapeutic agents.

We claim:
 1. A soluble recombinant protein complex comprising a firstpolypeptide covalently or non-covalently associated with a secondpolypeptide, wherein: a. the first polypeptide comprises the amino acidsequence of a first member of an interaction pair and an amino acidsequence that is at least 90% identical to amino acids 29-109 of SEQ IDNO: 1; wherein the first member of the interaction pair comprises afirst constant domain from an immunoglobulin; wherein the firstpolypeptide comprises a leucine at the amino acid position correspondingto position 79 of SEQ ID NO: 1; and b. the second polypeptide comprisesthe amino acid sequence of a second member of the interaction pair,wherein the second member of the interaction pair comprises a secondconstant domain from an immunoglobulin, and wherein the secondpolypeptide does not comprise a TGFβ superfamily type I or type IIreceptor polypeptide; and wherein the protein complex binds to GDF8and/or GDF11.
 2. The protein complex of claim 1, wherein the firstpolypeptide comprises, consists, or consists essentially of an aminoacid sequence that is: a. at least 95% identical to the sequence of SEQID No: 1; or b. at least 90% identical to a polypeptide that begins atany one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQID NO: 1, and ends at any one of amino acids 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:
 1. 3. The proteincomplex of claim 1, wherein the first constant domain from animmunoglobulin comprises an Fc immunoglobulin domain of IgG1immunoglobulin.
 4. The protein complex of claim 1, wherein the secondconstant domain from an immunoglobulin comprises an Fc immunoglobulindomain of IgG1 immunoglobulin.
 5. The protein complex of claim 1,wherein the first polypeptide and/or second polypeptide comprises one ormore modified amino acid residues selected from: a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, and an amino acid conjugated to an organic derivatizingagent.
 6. The protein complex of claim 1, wherein the first polypeptideand/or second polypeptide is glycosylated and has a glycosylationpattern obtainable from expression of the first polypeptide and/orsecond polypeptide in a CHO cell.
 7. The protein complex of claim 1,wherein the protein complex has one or more of the followingcharacteristics: i) binds to GDF8 and/or GDF11 with a KD of less than orequal to 10⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰ M; and ii) inhibits GDF8 and/orGDF11 receptor-mediated signaling transduction a cell.
 8. Apharmaceutical preparation comprising the protein complex of claim 1 anda pharmaceutically acceptable carrier.
 9. The protein complex of claim1, wherein the first polypeptide comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO: 106; and wherein the secondpolypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO:
 139. 10. The protein complex of claim 1, whereinthe first polypeptide comprises an amino acid sequence that is at least95% identical to SEQ ID NO: 106; and wherein the second polypeptidecomprises an amino acid sequence that is at least 95% identical to SEQID NO:
 139. 11. The protein complex of claim 1, wherein the firstpolypeptide comprises the amino acid sequence of SEQ ID NO: 106; andwherein the second polypeptide comprises the amino acid sequence of SEQID NO:
 139. 12. The protein complex of claim 1, wherein the firstpolypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 404; and wherein the second polypeptidecomprises an amino acid sequence that is at least 90% identical to SEQID NO:
 426. 13. The protein complex of claim 1, wherein the firstpolypeptide comprises an amino acid sequence that is at least 95%identical to SEQ ID NO: 404; and wherein the second polypeptidecomprises an amino acid sequence that is at least 95% identical to SEQID NO:
 426. 14. The protein complex of claim 1, wherein the firstpolypeptide comprises the amino acid sequence of SEQ ID NO: 404; andwherein the second polypeptide comprises the amino acid sequence of SEQID NO:
 426. 15. The protein complex of claim 1, wherein the firstpolypeptide comprises the amino acid sequence of a first member of aninteraction pair and an amino acid sequence that is at least 95%identical to amino acids 29-109 of SEQ ID NO:
 1. 16. The protein complexof claim 1, wherein the first polypeptide comprises the amino acidsequence of a first member of an interaction pair and an amino acidsequence that is identical to amino acids 29-109 of SEQ ID NO: 1.